mRNA stability and localization.RNA is critical at many stages of gene expression. How frequently it will be translated, how long it is likely to survive, and where in the cell it will be translated. RNA cis-elements & associated proteins
This document summarizes post-transcriptional modifications in eukaryotes. It discusses how eukaryotic mRNA undergoes processing, including capping, splicing to remove introns, and polyadenylation. Splicing requires snRNPs and the spliceosome to recognize splice sites. Alternative splicing allows one gene to code for multiple proteins. tRNA and rRNA also undergo processing as they mature, including modification of bases and removal of sequences. Final mature mRNA, tRNA, and rRNA are then ready for translation.
This document provides an overview of the origins and mechanisms of microRNAs (miRNAs) and small interfering RNAs (siRNAs). It discusses how double-stranded RNAs are cut by the enzyme Dicer into short RNA fragments that then base pair with mRNAs to induce degradation or transcriptional silencing. Key players in this RNA interference (RNAi) pathway include Dicer, Argonaute proteins, and the RNA-induced silencing complex (RISC). The document contrasts siRNAs, which originate from long double-stranded RNA, and miRNAs, which are encoded from single-stranded RNA precursors that form hairpin structures. It examines the processing steps and roles of various proteins in mediating the effects of si
This document discusses RNA interference (RNAi) and its mechanisms. It can be summarized as follows:
1. RNAi is a process where double-stranded RNA causes degradation of homologous mRNA sequences. It was discovered in 1998 and is found across many organisms.
2. The RNAi pathway involves conversion of dsRNA to siRNAs by the enzyme Dicer. siRNAs are incorporated into the RISC complex containing Argonaute proteins. RISC then cleaves and destroys homologous mRNA targets.
3. miRNAs are endogenous single-stranded RNAs that regulate gene expression at the translation level by preventing ribosome binding. They are processed from hairpin precursors by the enzymes Dro
This document provides an overview of non-coding RNA (ncRNA), including long ncRNAs and small ncRNAs. It discusses different types of small ncRNAs such as microRNAs (miRNAs), piRNAs, and small interfering RNAs (siRNAs). The document describes the biogenesis and mechanisms of action of these ncRNAs. It provides examples of functions for long ncRNAs and the role of the siRNA pathway in preventing transgenerational retrotransposition in plants under stress. In summary, the document categorizes and explains the major types and roles of coding and non-coding RNAs.
rRNA anr tRNA post transcriptional modificationsSidra Shaffique
Ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs) undergo processing in both prokaryotes and eukaryotes after their transcription. In E. coli, the primary rRNA transcripts are cleaved by endonucleases to produce pre-rRNAs, which are then trimmed to produce the mature rRNAs. In eukaryotes, snoRNAs direct site-specific methylation of the large primary rRNA transcript. tRNAs have their extra nucleotides removed and a conserved CCA trinucleotide added to their 3' ends by CCA-adding polymerase.
This document discusses long non-coding RNAs (lncRNAs). It begins by describing the discovery of lncRNAs in the 1980s-2000s through cDNA sequencing. It then states that lncRNAs are the largest class of transcripts in mouse and human genomes. The document discusses that lncRNAs were once thought to be useless but are now known to have regulatory functions. It provides details on the characteristics, locations in the genome, functions, mechanisms of action, roles in human disease, and implications in human carcinomas of lncRNAs.
RNA splicing is a process where introns are removed from precursor messenger RNA (pre-mRNA) and exons are joined together to produce mature mRNA. It occurs in the nucleus and is essential for eukaryotes to produce proteins. The spliceosome, a large complex of RNA and proteins, facilitates two transesterification reactions that remove introns and ligate exons. RNA splicing generates protein diversity through alternative splicing and is important for cellular functions and disease processes.
This document summarizes post-transcriptional modifications in eukaryotes. It discusses how eukaryotic mRNA undergoes processing, including capping, splicing to remove introns, and polyadenylation. Splicing requires snRNPs and the spliceosome to recognize splice sites. Alternative splicing allows one gene to code for multiple proteins. tRNA and rRNA also undergo processing as they mature, including modification of bases and removal of sequences. Final mature mRNA, tRNA, and rRNA are then ready for translation.
This document provides an overview of the origins and mechanisms of microRNAs (miRNAs) and small interfering RNAs (siRNAs). It discusses how double-stranded RNAs are cut by the enzyme Dicer into short RNA fragments that then base pair with mRNAs to induce degradation or transcriptional silencing. Key players in this RNA interference (RNAi) pathway include Dicer, Argonaute proteins, and the RNA-induced silencing complex (RISC). The document contrasts siRNAs, which originate from long double-stranded RNA, and miRNAs, which are encoded from single-stranded RNA precursors that form hairpin structures. It examines the processing steps and roles of various proteins in mediating the effects of si
This document discusses RNA interference (RNAi) and its mechanisms. It can be summarized as follows:
1. RNAi is a process where double-stranded RNA causes degradation of homologous mRNA sequences. It was discovered in 1998 and is found across many organisms.
2. The RNAi pathway involves conversion of dsRNA to siRNAs by the enzyme Dicer. siRNAs are incorporated into the RISC complex containing Argonaute proteins. RISC then cleaves and destroys homologous mRNA targets.
3. miRNAs are endogenous single-stranded RNAs that regulate gene expression at the translation level by preventing ribosome binding. They are processed from hairpin precursors by the enzymes Dro
This document provides an overview of non-coding RNA (ncRNA), including long ncRNAs and small ncRNAs. It discusses different types of small ncRNAs such as microRNAs (miRNAs), piRNAs, and small interfering RNAs (siRNAs). The document describes the biogenesis and mechanisms of action of these ncRNAs. It provides examples of functions for long ncRNAs and the role of the siRNA pathway in preventing transgenerational retrotransposition in plants under stress. In summary, the document categorizes and explains the major types and roles of coding and non-coding RNAs.
rRNA anr tRNA post transcriptional modificationsSidra Shaffique
Ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs) undergo processing in both prokaryotes and eukaryotes after their transcription. In E. coli, the primary rRNA transcripts are cleaved by endonucleases to produce pre-rRNAs, which are then trimmed to produce the mature rRNAs. In eukaryotes, snoRNAs direct site-specific methylation of the large primary rRNA transcript. tRNAs have their extra nucleotides removed and a conserved CCA trinucleotide added to their 3' ends by CCA-adding polymerase.
This document discusses long non-coding RNAs (lncRNAs). It begins by describing the discovery of lncRNAs in the 1980s-2000s through cDNA sequencing. It then states that lncRNAs are the largest class of transcripts in mouse and human genomes. The document discusses that lncRNAs were once thought to be useless but are now known to have regulatory functions. It provides details on the characteristics, locations in the genome, functions, mechanisms of action, roles in human disease, and implications in human carcinomas of lncRNAs.
RNA splicing is a process where introns are removed from precursor messenger RNA (pre-mRNA) and exons are joined together to produce mature mRNA. It occurs in the nucleus and is essential for eukaryotes to produce proteins. The spliceosome, a large complex of RNA and proteins, facilitates two transesterification reactions that remove introns and ligate exons. RNA splicing generates protein diversity through alternative splicing and is important for cellular functions and disease processes.
RNA serves various essential functions in biology. There are two main types: coding RNA (mRNA) which is translated into proteins, and non-coding RNA (ncRNA) which has regulatory functions but is not translated. Major ncRNAs include tRNA, which transports amino acids to the ribosome during protein synthesis, and rRNA, which along with proteins makes up the ribosome and catalyzes peptide bond formation. NcRNAs can regulate genes at the transcription or translation level. In eukaryotes, the majority of genomic transcripts are ncRNAs with diverse roles in splicing, translation, and gene expression control.
RNA polymerase is an enzyme that produces RNA in cells. It was discovered in 1960 and is essential for all organisms. In prokaryotes, a single RNA polymerase synthesizes different RNA types, while eukaryotic RNA polymerase is a multi-subunit enzyme. RNA polymerase I synthesizes rRNA for ribosomes, polymerase II synthesizes pre-mRNA and most snRNA/miRNA, and polymerase III synthesizes tRNA and other small RNAs. The transcription process involves initiation, elongation, and termination stages.
transcription activators, repressors, & control RNA splicing, procesing and e...ranjithahb ranjithahbhb
RNA processing involves several steps to convert primary transcripts into mature mRNA in eukaryotic cells. These include 5' capping, 3' cleavage and polyadenylation, and RNA splicing. RNA splicing involves two transesterification reactions that remove introns and join exons. Alternative splicing allows a single gene to produce multiple protein variants. Eukaryotic gene expression is regulated by transcriptional activators and repressors that bind cis-regulatory elements like promoters and enhancers. Activators recruit transcriptional machinery while repressors inhibit transcription. Chromatin structure also influences transcription with acetylation associated with active genes.
This document discusses different strategies for cloning DNA fragments from complex sources like genomic DNA or cDNA. There are two major approaches - cell-based cloning, which divides the DNA into fragments that are cloned to create a library, and directly amplifying target sequences using PCR. The document focuses on cDNA library construction, explaining that cDNA libraries reveal gene expression profiles. It describes early cDNA cloning methods and their limitations, as well as improved directional and non-directional cloning techniques. Finally, it discusses various screening methods for identifying clones of interest from cDNA libraries, including colony hybridization, plaque lifts and immunological screening.
Eukaryotic translation is the process by which messenger RNA is translated into proteins. It involves three main phases: initiation, elongation, and termination. Initiation requires several eukaryotic initiation factors to form a pre-initiation complex and recruit the small ribosomal subunit to the 5' end of mRNA. Elongation then adds amino acids to the growing polypeptide chain via three elongation factors. Termination occurs when release factors recognize a stop codon and allow dissociation of the ribosome and release of the completed protein. The process is more complex in eukaryotes compared to prokaryotes due to the larger ribosome size and additional initiation factors required.
TATA binding proteins (TBPs) play an essential role in eukaryotic transcription. TBP is a subunit of the general transcription factor TFIID that binds to the TATA box upstream of core promoters. TBP binds in the minor groove of DNA and bends it into an 80 degree curve. TBP adopts a saddle-shaped structure that positions the concave surface to interact with DNA while exposing the convex surface to recruit other general transcription factors and form the preinitiation complex. TBP is universally required for transcription by all three eukaryotic RNA polymerases and some genes in Archaea as well.
RNA splicing is the process by which introns, or non-coding sequences, are removed from pre-messenger RNA (pre-mRNA) to produce mature mRNA that can be translated into protein. Most genes contain introns that are removed by a spliceosome, a complex of RNA and proteins, leaving just the coding exons to form mRNA. Alternative splicing allows one gene to encode multiple proteins by selecting different combinations of exons. Errors in splicing can cause diseases if they result in truncated or abnormal proteins.
The S1 nuclease was extracted from Aspergill suoryzae. The S1 nuclease is a specific
single-stranded endonuclease. It can degrade single-stranded DNA and
single-stranded RNA to produce 5'-single-stranded nucleotides or oligonucleotides.
The document discusses transcription in prokaryotes and eukaryotes. In prokaryotes, RNA polymerase binds to promoter sequences and transcribes DNA into RNA through initiation, elongation, and termination. Transcription requires RNA polymerase and proceeds similarly in eukaryotes but involves multiple RNA polymerases and occurs in the nucleus. Eukaryotic transcription is more complex, utilizing regulatory sequences, transcription factors, and RNA processing to modify pre-mRNA into mature mRNA through splicing, capping, polyadenylation, and other modifications. Mutations can affect splicing and cause genetic disorders like beta-thalassemia.
Arabinose Operon is a self-regulatory sequence of genes used by material to metabolize a five-carbon sugar called arabinose when there is a deficiency of glucose in the environment.
Dna supercoiling and role of topoisomerasesYashwanth B S
supercoiling is one of the important process to condenses the huge amount of DNA to fit inside the histone and its also plays a role during the replication ,transcription etc..,these activities is carried out by an enzyme called topoisomerases.
The document discusses the basic principles of gene expression from DNA to protein. It describes transcription, which is the synthesis of RNA from a DNA template, and translation, which is the synthesis of proteins from mRNA templates using ribosomes. In eukaryotes, transcription requires RNA polymerases and other transcription factors to initiate transcription from DNA. The primary transcript then undergoes processing including 5' capping, 3' polyadenylation, and splicing to form mature mRNA. The mRNA is then translated by ribosomes to produce proteins.
RNA splicing is a process in which introns are removed from pre-mRNA transcripts and exons are joined together to produce mature mRNA. There are three main types of splicing pathways: spliceosomal splicing, self-splicing, and tRNA splicing. Spliceosomal splicing involves the spliceosome complex and is the most common in eukaryotes. Self-splicing occurs without proteins through ribozyme activity. tRNA splicing uses ribonucleases and ligases. Alternative splicing allows different mRNA isoforms to be produced from the same pre-mRNA. Splicing errors can cause genetic diseases by disrupting protein sequences.
Current trends in pseduogene detection and characterizationShreya Feliz
This presentation gives the insight of the current trends in detecting and characterizing Pseudogenes. Pseudogenes detection by bioinformatics may enhance the understanding of Pseudogenes and take research to the next step.
A gene library is a large collection of DNA fragments cloned from an organism. It contains genomic DNA or cDNA sequences. Gene libraries are constructed using molecular tools like restriction enzymes and ligases to cut and paste DNA fragments into vectors such as plasmids, phages, or artificial chromosomes. The choice of vector depends on the size of the genome being cloned. Libraries allow screening to identify genes of interest through techniques like hybridization or expression screening. cDNA libraries contain only expressed sequences without introns, making them preferable for cloning eukaryotic genes in prokaryotes.
The document discusses the process of synthesizing cDNA from mRNA. It involves isolating mRNA, using reverse transcriptase to copy the mRNA into single-stranded cDNA, then converting it to double-stranded cDNA using DNA polymerase. The double-stranded cDNA can then be inserted into a vector and used to create a cDNA library through cloning in bacteria or phage. The library can be screened by hybridization or assays to identify clones containing genes of interest.
This document discusses nucleic acid probes and their use in hybridization experiments. It notes that probes are short sequences of nucleotides that bind to specific target sequences. The degree of homology between the probe and target determines how stable the hybridization is. Probes can range in size from 10 to over 10,000 nucleotide bases, with most common probes being 14 to 40 bases. Short probes hybridize quickly but have less specificity, while longer probes hybridize more stably. The document then describes different methods for labeling probes, including nick translation, primer extension, RNA polymerase transcription, end-labeling, and direct labeling. It also discusses factors that affect probe specificity and hybridization conditions.
Post-transcriptional modifications help process primary transcripts into mRNA in three main ways: 1) 5' capping protects the transcript and aids export from the nucleus, 2) Polyadenylation aids stability and transport, and 3) Splicing removes introns and ligates exons to form mature mRNA. In eukaryotes, this occurs in the nucleus and is essential for efficient translation. It can also result in alternative splicing to increase protein diversity from a single gene.
DNA Protein interaction occur when a protein binds a molecule of DNA, often to regulate the biological function of DNA, usually the expression of a gene. DNA Protein interactions play very vital roles in any living cell. It controls various cellular processes which are very essential for living beings, viz. replication, transcription, recombination, DNA repair etc. There are several types of proteins found in a cell.Direct recognition occurs when the amino acid side chains of a protein interact with specific DNA bases.
Most protein-DNA interactions are mediated by direct physical interaction (hydrogen bonding or hydrophobic interactions) between the protein and the DNA base pairs.
DNA-binding proteins can be identified by many experimental techniques such as chromatin immunoprecipitation on microarrays, X-ray crystallography and nuclear magnetic resonance (NMR).
L10. enzymes used in genetic engineering i-1Rishabh Jain
This document discusses various enzymes that are used in genetic engineering and recombinant DNA technology. It describes DNA and RNA polymerases such as DNA polymerase I, Klenow fragment, T4 DNA polymerase, and reverse transcriptase. It also covers ligases, phosphatases, kinases, and nucleases including DNase I, and their functions, sources, and applications in techniques like cDNA synthesis, DNA labeling, amplification, and sequencing.
Development biology (rna processing and translational regulation of developme...mehwishkhan78
Cellular differentiation occurs through gene expression and protein production regulated by RNA processing and translation mechanisms. There are two major ways that differential RNA processing regulates development: 1) Through splicing, where one gene can create different proteins from alternative combinations of exons. 2) Through "censoring" or selecting different nuclear transcripts to be processed into cytoplasmic mRNA in different cell types. Translational regulation also controls developmental processes by mechanisms like phosphorylation of initiation factors that turn translation on and off.
types and structure of prokaryotic RNATooba Kanwal
RNA exists in different single-stranded structures that are involved in protein synthesis or regulation. Messenger RNA (mRNA) carries genetic information from DNA to the ribosome. Ribosomal RNA (rRNA) is a component of ribosomes and facilitates protein translation. Transfer RNA (tRNA) transports amino acids to the ribosome and translates mRNA codons into amino acids during protein synthesis.
RNA serves various essential functions in biology. There are two main types: coding RNA (mRNA) which is translated into proteins, and non-coding RNA (ncRNA) which has regulatory functions but is not translated. Major ncRNAs include tRNA, which transports amino acids to the ribosome during protein synthesis, and rRNA, which along with proteins makes up the ribosome and catalyzes peptide bond formation. NcRNAs can regulate genes at the transcription or translation level. In eukaryotes, the majority of genomic transcripts are ncRNAs with diverse roles in splicing, translation, and gene expression control.
RNA polymerase is an enzyme that produces RNA in cells. It was discovered in 1960 and is essential for all organisms. In prokaryotes, a single RNA polymerase synthesizes different RNA types, while eukaryotic RNA polymerase is a multi-subunit enzyme. RNA polymerase I synthesizes rRNA for ribosomes, polymerase II synthesizes pre-mRNA and most snRNA/miRNA, and polymerase III synthesizes tRNA and other small RNAs. The transcription process involves initiation, elongation, and termination stages.
transcription activators, repressors, & control RNA splicing, procesing and e...ranjithahb ranjithahbhb
RNA processing involves several steps to convert primary transcripts into mature mRNA in eukaryotic cells. These include 5' capping, 3' cleavage and polyadenylation, and RNA splicing. RNA splicing involves two transesterification reactions that remove introns and join exons. Alternative splicing allows a single gene to produce multiple protein variants. Eukaryotic gene expression is regulated by transcriptional activators and repressors that bind cis-regulatory elements like promoters and enhancers. Activators recruit transcriptional machinery while repressors inhibit transcription. Chromatin structure also influences transcription with acetylation associated with active genes.
This document discusses different strategies for cloning DNA fragments from complex sources like genomic DNA or cDNA. There are two major approaches - cell-based cloning, which divides the DNA into fragments that are cloned to create a library, and directly amplifying target sequences using PCR. The document focuses on cDNA library construction, explaining that cDNA libraries reveal gene expression profiles. It describes early cDNA cloning methods and their limitations, as well as improved directional and non-directional cloning techniques. Finally, it discusses various screening methods for identifying clones of interest from cDNA libraries, including colony hybridization, plaque lifts and immunological screening.
Eukaryotic translation is the process by which messenger RNA is translated into proteins. It involves three main phases: initiation, elongation, and termination. Initiation requires several eukaryotic initiation factors to form a pre-initiation complex and recruit the small ribosomal subunit to the 5' end of mRNA. Elongation then adds amino acids to the growing polypeptide chain via three elongation factors. Termination occurs when release factors recognize a stop codon and allow dissociation of the ribosome and release of the completed protein. The process is more complex in eukaryotes compared to prokaryotes due to the larger ribosome size and additional initiation factors required.
TATA binding proteins (TBPs) play an essential role in eukaryotic transcription. TBP is a subunit of the general transcription factor TFIID that binds to the TATA box upstream of core promoters. TBP binds in the minor groove of DNA and bends it into an 80 degree curve. TBP adopts a saddle-shaped structure that positions the concave surface to interact with DNA while exposing the convex surface to recruit other general transcription factors and form the preinitiation complex. TBP is universally required for transcription by all three eukaryotic RNA polymerases and some genes in Archaea as well.
RNA splicing is the process by which introns, or non-coding sequences, are removed from pre-messenger RNA (pre-mRNA) to produce mature mRNA that can be translated into protein. Most genes contain introns that are removed by a spliceosome, a complex of RNA and proteins, leaving just the coding exons to form mRNA. Alternative splicing allows one gene to encode multiple proteins by selecting different combinations of exons. Errors in splicing can cause diseases if they result in truncated or abnormal proteins.
The S1 nuclease was extracted from Aspergill suoryzae. The S1 nuclease is a specific
single-stranded endonuclease. It can degrade single-stranded DNA and
single-stranded RNA to produce 5'-single-stranded nucleotides or oligonucleotides.
The document discusses transcription in prokaryotes and eukaryotes. In prokaryotes, RNA polymerase binds to promoter sequences and transcribes DNA into RNA through initiation, elongation, and termination. Transcription requires RNA polymerase and proceeds similarly in eukaryotes but involves multiple RNA polymerases and occurs in the nucleus. Eukaryotic transcription is more complex, utilizing regulatory sequences, transcription factors, and RNA processing to modify pre-mRNA into mature mRNA through splicing, capping, polyadenylation, and other modifications. Mutations can affect splicing and cause genetic disorders like beta-thalassemia.
Arabinose Operon is a self-regulatory sequence of genes used by material to metabolize a five-carbon sugar called arabinose when there is a deficiency of glucose in the environment.
Dna supercoiling and role of topoisomerasesYashwanth B S
supercoiling is one of the important process to condenses the huge amount of DNA to fit inside the histone and its also plays a role during the replication ,transcription etc..,these activities is carried out by an enzyme called topoisomerases.
The document discusses the basic principles of gene expression from DNA to protein. It describes transcription, which is the synthesis of RNA from a DNA template, and translation, which is the synthesis of proteins from mRNA templates using ribosomes. In eukaryotes, transcription requires RNA polymerases and other transcription factors to initiate transcription from DNA. The primary transcript then undergoes processing including 5' capping, 3' polyadenylation, and splicing to form mature mRNA. The mRNA is then translated by ribosomes to produce proteins.
RNA splicing is a process in which introns are removed from pre-mRNA transcripts and exons are joined together to produce mature mRNA. There are three main types of splicing pathways: spliceosomal splicing, self-splicing, and tRNA splicing. Spliceosomal splicing involves the spliceosome complex and is the most common in eukaryotes. Self-splicing occurs without proteins through ribozyme activity. tRNA splicing uses ribonucleases and ligases. Alternative splicing allows different mRNA isoforms to be produced from the same pre-mRNA. Splicing errors can cause genetic diseases by disrupting protein sequences.
Current trends in pseduogene detection and characterizationShreya Feliz
This presentation gives the insight of the current trends in detecting and characterizing Pseudogenes. Pseudogenes detection by bioinformatics may enhance the understanding of Pseudogenes and take research to the next step.
A gene library is a large collection of DNA fragments cloned from an organism. It contains genomic DNA or cDNA sequences. Gene libraries are constructed using molecular tools like restriction enzymes and ligases to cut and paste DNA fragments into vectors such as plasmids, phages, or artificial chromosomes. The choice of vector depends on the size of the genome being cloned. Libraries allow screening to identify genes of interest through techniques like hybridization or expression screening. cDNA libraries contain only expressed sequences without introns, making them preferable for cloning eukaryotic genes in prokaryotes.
The document discusses the process of synthesizing cDNA from mRNA. It involves isolating mRNA, using reverse transcriptase to copy the mRNA into single-stranded cDNA, then converting it to double-stranded cDNA using DNA polymerase. The double-stranded cDNA can then be inserted into a vector and used to create a cDNA library through cloning in bacteria or phage. The library can be screened by hybridization or assays to identify clones containing genes of interest.
This document discusses nucleic acid probes and their use in hybridization experiments. It notes that probes are short sequences of nucleotides that bind to specific target sequences. The degree of homology between the probe and target determines how stable the hybridization is. Probes can range in size from 10 to over 10,000 nucleotide bases, with most common probes being 14 to 40 bases. Short probes hybridize quickly but have less specificity, while longer probes hybridize more stably. The document then describes different methods for labeling probes, including nick translation, primer extension, RNA polymerase transcription, end-labeling, and direct labeling. It also discusses factors that affect probe specificity and hybridization conditions.
Post-transcriptional modifications help process primary transcripts into mRNA in three main ways: 1) 5' capping protects the transcript and aids export from the nucleus, 2) Polyadenylation aids stability and transport, and 3) Splicing removes introns and ligates exons to form mature mRNA. In eukaryotes, this occurs in the nucleus and is essential for efficient translation. It can also result in alternative splicing to increase protein diversity from a single gene.
DNA Protein interaction occur when a protein binds a molecule of DNA, often to regulate the biological function of DNA, usually the expression of a gene. DNA Protein interactions play very vital roles in any living cell. It controls various cellular processes which are very essential for living beings, viz. replication, transcription, recombination, DNA repair etc. There are several types of proteins found in a cell.Direct recognition occurs when the amino acid side chains of a protein interact with specific DNA bases.
Most protein-DNA interactions are mediated by direct physical interaction (hydrogen bonding or hydrophobic interactions) between the protein and the DNA base pairs.
DNA-binding proteins can be identified by many experimental techniques such as chromatin immunoprecipitation on microarrays, X-ray crystallography and nuclear magnetic resonance (NMR).
L10. enzymes used in genetic engineering i-1Rishabh Jain
This document discusses various enzymes that are used in genetic engineering and recombinant DNA technology. It describes DNA and RNA polymerases such as DNA polymerase I, Klenow fragment, T4 DNA polymerase, and reverse transcriptase. It also covers ligases, phosphatases, kinases, and nucleases including DNase I, and their functions, sources, and applications in techniques like cDNA synthesis, DNA labeling, amplification, and sequencing.
Development biology (rna processing and translational regulation of developme...mehwishkhan78
Cellular differentiation occurs through gene expression and protein production regulated by RNA processing and translation mechanisms. There are two major ways that differential RNA processing regulates development: 1) Through splicing, where one gene can create different proteins from alternative combinations of exons. 2) Through "censoring" or selecting different nuclear transcripts to be processed into cytoplasmic mRNA in different cell types. Translational regulation also controls developmental processes by mechanisms like phosphorylation of initiation factors that turn translation on and off.
types and structure of prokaryotic RNATooba Kanwal
RNA exists in different single-stranded structures that are involved in protein synthesis or regulation. Messenger RNA (mRNA) carries genetic information from DNA to the ribosome. Ribosomal RNA (rRNA) is a component of ribosomes and facilitates protein translation. Transfer RNA (tRNA) transports amino acids to the ribosome and translates mRNA codons into amino acids during protein synthesis.
The document summarizes key concepts about transcription and translation. It defines transcription as copying genetic information from DNA to mRNA. Translation is defined as mRNA being used as a template to synthesize a protein through the genetic code. The three main steps of translation are: 1) Transfer RNA transports amino acids, 2) mRNA and the genetic code are used to match codons to amino acids, 3) Ribosomes assemble proteins using mRNA and tRNAs.
This document provides an overview of microbial genetics. It discusses the levels of genetic structure including genomes, chromosomes, and genes. It describes the structure and replication of DNA, as well as transcription and translation processes. Key concepts covered include DNA structure, semi-conservative replication, the central dogma of molecular biology, transcription, translation, and gene regulation via operons such as the lactose and arginine operons. The document also briefly discusses mutations, DNA repair mechanisms, horizontal gene transfer processes, and transposons.
This document summarizes key aspects of gene transcription including:
1. Transcription is important for regulating cellular function and aberrant control can cause disease.
2. In eukaryotes, transcription and translation are separated in space and time, and primary RNA transcripts undergo extensive processing.
3. Prokaryotic transcription involves RNA polymerase recognizing promoters and transcribing DNA into RNA with sigma factors providing specificity. Eukaryotic transcription involves three RNA polymerases and more complex promoters.
- mRNA carries genetic information from DNA to the ribosome where protein synthesis occurs. It is single-stranded and complementary to one DNA strand.
- rRNA makes up 80% of cellular RNA and is a component of ribosomes. rRNA molecules synthesized in the nucleolus combine with proteins to form ribosomes, the sites of protein synthesis.
- tRNA acts as an intermediary between mRNA and amino acids during protein synthesis. It has a cloverleaf secondary structure and binds to specific amino acids to deliver them to the ribosome based on the mRNA sequence.
The central dogma describes how DNA is transcribed into mRNA which is then translated into protein. There are three eukaryotic RNA polymerases that transcribe different types of RNA. Polymerase I transcribes rRNA, polymerase II transcribes mRNA and small RNAs, and polymerase III transcribes tRNA. Transcription occurs when mRNA is produced from DNA, with thymine in DNA pairing with uracil in mRNA. Translation then occurs as each mRNA codon codes for a specific amino acid, with the ribosome facilitating the process to produce the protein based on the mRNA sequence. Gene expression is regulated at multiple steps including transcription, translation, and protein turnover through transcription factors that turn genes on and off.
The central dogma of molecular biology describes how DNA is transcribed into RNA and then translated into protein. There are three eukaryotic RNA polymerases that transcribe different types of RNA. Polymerase I transcribes ribosomal RNA, polymerase II transcribes messenger RNA and some small RNAs, and polymerase III transcribes transfer RNA. Transcription occurs when DNA is copied into messenger RNA, which then undergoes processing before being exported from the nucleus for translation. Translation is the process by which the codons of messenger RNA are used to specify the amino acid sequence of proteins with the help of transfer RNA and ribosomes. Gene expression is regulated at multiple steps including transcription, translation, and protein turnover by transcription
Anatomy and Physiology RNA and Proteinsmrhunterspage
Gene expression is the process by which the information from a gene is used in the synthesis of a functional gene product. There are two main types of RNA - messenger RNA and transfer RNA - that are involved in reading instructions from DNA and assembling amino acids to make proteins. The process of gene expression involves transcription of DNA into mRNA in the cell nucleus, which is then read by ribosomes during translation to produce proteins from the instructions provided in the mRNA.
Junk DNA/ Non-coding DNA and its Importance (Regulatory RNAs, RNA interferen...Pradeep Singh Narwat
The document discusses various types of non-coding DNA sequences, including repetitive sequences, transposons, non-coding RNAs, introns, and pseudogenes. It notes that while genes only make up 2-3% of human DNA, recent projects like ENCODE have found that a much larger portion of non-coding DNA is functionally important, for example through transcriptional and translational regulation of protein-coding sequences. The document outlines different classes of transposons, introns, non-coding RNAs and their various roles in gene expression, epigenetics, and genome evolution.
Differential RNA processing regulates animal development through nuclear RNA selection and differential RNA splicing. Nuclear RNA selection involves selecting which RNA transcripts get exported from the nucleus and translated, allowing different cell types to produce only necessary proteins. Differential RNA splicing allows a single gene to code for multiple proteins through varying combinations of exons, done by splicing different exons together to produce distinct mRNAs and proteins. This mechanism generates splicing isoforms and is regulated by spliceosomes, snRNPs, and splicing factors that recognize exons and introns.
Expression of genetic material : From Transcription to TranslationSreshti Bagati
This document summarizes key aspects of gene expression from transcription to translation in eukaryotes. It describes the central dogma principle where genetic information flows from DNA to RNA to proteins. Transcription involves RNA polymerase and various transcription factors. The mRNA produced undergoes processing including capping, polyadenylation, and splicing before translation. Translation occurs on ribosomes and involves initiation, elongation and termination factors to produce proteins.
Transcription is the process by which messenger RNA (mRNA) is produced from DNA. RNA polymerase adds RNA nucleotides to the growing mRNA molecule in a 5' to 3' direction. Gene expression during transcription is regulated by nucleosomes, DNA methylation, promoter sequences, regulatory proteins, and environmental factors. After transcription, mRNA contains both exons that are expressed as proteins during translation and introns that are removed by splicing. Splicing can change the number of exons and produce different polypeptides.
The document discusses post-transcriptional modifications that occur in eukaryotic mRNA. It describes three major modifications: 1) 5' capping, which adds a 7-methylguanosine cap to protect the mRNA and enhance translation, 2) 3' polyadenylation, which adds a poly-A tail to stabilize the mRNA and influence translation and stability, and 3) splicing, by which introns are removed from pre-mRNA through the spliceosome complex to produce mature mRNA for translation.
Covers the flow of information from DNA to Protein synthesis, Transcription, Types of RNA, Genetic code, Protein Synthesis, Cell Function and cell reproduction
This document discusses genetic control and regulation at the molecular level. It covers DNA and RNA structure, transcription, translation, gene regulation, and cell division. The central dogma of molecular genetics is described involving DNA transcription to RNA and translation to protein. The types of RNA are defined including mRNA, tRNA, rRNA. Transcription and translation processes are explained in detail. Gene regulation mechanisms like promoters, transcription factors, and feedback systems are covered. Finally, the document discusses cell reproduction through DNA replication and cell mitosis.
The document describes the genetic control of protein synthesis. It discusses how genes in the cell nucleus control protein synthesis through the processes of transcription and translation. There are approximately 30,000 genes in each cell, each composed of DNA. During transcription, the DNA code is transferred to an RNA code, which then directs protein synthesis. The main types of RNA - mRNA, tRNA, rRNA and miRNA - are described along with their roles in protein synthesis. Translation and the genetic code are also summarized.
This document provides an overview of molecular biology concepts including:
- DNA contains genetic codes that are identical in all cells of an organism. The genome is the entire DNA complement and controls protein production.
- DNA is made up of nucleotides that form base pairs (A-T, C-G) along a phosphate-sugar backbone forming two complementary strands that twist into the double helix structure.
- Genes are portions of DNA that are transcribed into messenger RNA (mRNA), which is then translated into proteins according to its nucleotide sequence using codons.
CRISPR is a powerful new tool for genome editing that allows targeted modifications to genes. It utilizes the Cas9 enzyme to cut DNA at a specific site guided by a short RNA molecule. This summary will discuss the history and mechanisms of CRISPR/Cas9 and its applications in biotechnology and agriculture. CRISPR represents a major breakthrough that will revolutionize genetic engineering by enabling precise edits to genomes. However, further refinement is needed to address issues such as off-target effects. Overall, CRISPR technology holds tremendous promise for developing improved crop traits.
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it describes the bony anatomy including the femoral head , acetabulum, labrum . also discusses the capsule , ligaments . muscle that act on the hip joint and the range of motion are outlined. factors affecting hip joint stability and weight transmission through the joint are summarized.
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2. 1. Introduction
•RNA is critical at many stages of gene expression
•Intermediate in protein synthesis
•How frequently it will be translated, how long it is likely to survive,
and where in the cell it will be translated
•RNA cis-elements & associated proteins
2
3. Features of prokaryotic and eukaryotic mRNAs:
a) Bacterial mRNA
b) Eukaryotic mRNAs
c) Histone mRNAs
3
4. 2. Messenger RNAs Unstable Molecules
1) mRNA instability Action of ribonucleases
2) Ribonucleases differ in their substrate preference and mode
of attack
3) Half-lives
4) Differential mRNA stability
4
5. •Unstable molecules
•Ribonucleases: DNA replication, DNA repair, processing of new transcripts &
the degradation of mRNA.
1) Endoribonucleases
2) Exoribonucleases
5
7. 3. Eukaryotic mRNAs mRNPs
1) mRNA associates with population of proteins
2) mRNP proteins have roles in the cytoplasm
3) RNA-binding proteins uncharacterized
4) mRNAs overlapping : regulon
7
8. •Eukaryotic mRNAs RNPs
•Nuclear maturation: RNA sequence & complement of proteins + TREX EXIT
•Pioneering round : defective template destroyed
•The “nuclear history” of an mRNA is critical in determining its fate
in the cytoplasm.
•RBPs
•RNA regulon: The set of mRNAs
that share a particular type of RBP
8
9. 4. Prokaryotic mRNA Degradation Multiple
Enzymes
1) Initiated by removal of a pyrophosphate from the 5′ terminus
2) Monophosphorylated mRNAs two-step cycle
3) 3′ polyadenylation : facilitate the degradation
4) Main degradation enzymes : degradosome
9
10. •In prokaryotes: mRNA degradation occurs during the process of
coupled transcription/translation.
•Prokaryotic ribosomes begin translation even before transcription
is completed
•polyribosome or polysome
•Endonuclease + 3′ to 5′ exonuclease = mRNA degradation
10
11. •The major mRNA degradation pathway in E. coli is a multistage
process:
1) pyrophosphate from the 5′ terminus
single phosphate
Monophosphorylate RNaseE
cut near the 5′ end of the mRNA
monocistronic mRNA
2) PNPase(3′ to 5′ exonuclease) upstream fragment
• This two-step ribonuclease cycle is repeated along the
length of the mRNA
11
12. • PNPase are unable to progress through double-stranded regions = stem-loop structure at
the 3′ end mRNAs protects
• Degradosome RNase E (N-terminal: endonuclease / C-terminal: scaffold)
PNPase (degradation: endonuclease + exonuclease )
• Inactivation of RNase E mRNA degradation
• Mutations PNPase no effect on overall mRNA stability
• The half-lives of specific mRNAs can be altered by different cellular
physiological states such as starvation or other forms of stress.
12
13. 5. Degraded Two
Deadenylation-Dependent Pathways
1) Modifications at both ends of mRNA protect against
degradation
2) mRNA decay pathways Initiated by deadenylation
3) Deadenylation: decapping & exonuclease digestion(5′ or 3′)
4) The decapping enzyme competes with the translation initiation
complex (5′ cap binding)
5) Exosome mRNA digestion(3′)
6) Degradation PBs
13
14. •Eukaryotic mRNAs are protected from exonucleases by their
modified ends
•The histone mRNAs in mammals stem-loop structure
•Degradation of the vast majority of mRNAs is deadenylation
dependent
•In both yeast and mammalian cells, the poly(A) tail is initially
shortened by the PAN2/3 complex, followed by a more rapid
digestion of the remaining 60 to 80 A tail by a second complex,
CCR4-NOT, which contains the processive exonuclease CCR4
and at least eight other subunits. 14
15. • Two different mRNA degradation pathways:
1) digestion of the poly(A) tail decapping 5′ monophosphorylated exonuclease Xrn1
2) deadenylation poly(A) exonuclease digestion of the body of the mRNA exosome
15
16. 6. Other Degradation Pathways Target Specific
mRNAs
1) Four additional degradation pathways
2) Deadenylation-independent decapping poly(A) tail
3) degradation of histone mRNAs 3′ poly(U) tail
4) Degradation of some mRNAs sequence- or structure-
specific endonucleolytic
5) mRNAs are targeted for degradation or translational
repression by microRNAs 16
17. Four other pathways for mRNA degradation:
1) Deadenylation-independent decapping Xrn1
2) Histone mRNAs in mammalian Lsm1–7 , exosome
3) Endonucleotic cleavage digestion 3‘ or 5' decapping
4) microRNA(miRNA) endonucleotic cleavage, deadenylation,
translational repression
•It has been estimated that 50% of all mRNAs could be regulated by
miRNAs.
17
18. 18
Summary of key elements of mRNA
decay pathways in eukaryotic cells
19. • The rate of deadenylation and/or other steps in degradation by these pathways
can be controlled by cis-acting elements in each mRNA and trans-acting
factors present in the cell.
19
20. 7. mRNA Half-Lives Controlled by Sequences or
Structures Within the mRNA
1) cis-elements degradation
2) Destabilizing elements: mRNA decay , Stabilizing elements: mRNA decay
3) AU-rich elements(AREs) destabilizing elements in mammals
4) DE-binding proteins + decay machinery degradation
5) SEs stable mRNAs
6) mRNA degradation rates variety of signals
20
21. What accounts for the large range of half-lives of different mRNAs
in the same cell?
1) cis-elements 3′ UTR
2) Destabilizing elements (DEs)
3) AU-rich element (ARE) pentamer sequence AUUUA
4) Stabilizing elements (SEs) 3′ UTR
21
22. 22
• An example of regulated mRNA stabilization occurs for the mammalian transferrin mRNA
• IRE + pro 3′ UTR mRNA is stabilized
• IRE-binding protein mRNA is stabilized
• Microarray studies have shown that
almost 50% of changes in mRNA levels
stimulated by cellular signals are due to
mRNA stabilization or destabilization
events, not to Transcriptional changes.
23. 8. Newly Synthesized RNAs Nuclear Surveillance
System
1) Aberrant nuclear RNAs are identified and destroyed by a surveillance
system
2) Nuclear exosome: processing of normal substrate RNAs &
destruction of aberrant RNAs
3) TRAMP complex facilitated 3′ exonuclease activity
4) Exosome degradation unspliced or aberrantly spliced pre-mRNAs
5) CUTs destroyed in the nucleus 23
24. •All newly synthesized RNAs are subject to multiple processing
steps after they are transcribed
•Surveillance involves two kinds of activities:
1) Identify and tag the aberrant substrate RNA
2) Destroy it
•The destroyer is the nuclear exosome 3′ to 5′ exonuclease
1) RNA processing of some noncoding RNA transcripts: snRNA, snoRNA, and Rrna
2) degradation of aberrant transcripts
24
25. For example:
1) Nrd1–Nab3
2) TRAMP
What are the substrates for TRAMP–exosome degradation?
What kinds of abnormalities condemn pre-mRNAs to nuclear destruction?
1) Unspliced or aberrantly spliced pre-mRNAs
2) Improperly terminated, lacking a poly(A) tail.
25
26. •Whereas polyadenylation is protective in true mRNAs, it may
actually be destabilizing for cryptic unstable transcripts(CUTs).
•More than three-quarters of RNA Pol II transcripts may be composed
of noncoding RNAs and be subject to rapid degradation by the
exosome!
26
27. 9. Control of mRNA Translation : Cytoplasmic
Surveillance Systems
1) Nonsense-mediated decay (NMD): mRNAs with premature stop
codons
2) NMD: UPF and SMG proteins
3) Recognition of a termination codon: unusual 3′ UTR structure & EJCs
4) Nonstop decay (NSD): termination codon
5) No-go decay (NGD): mRNAs with stalled ribosomes in their coding
regions 27
28. •Some kinds of mRNA defects can be assessed only during translation
Substrates for cytoplasmic surveillance systems:
1) Nonsense-mediated decay (NMD)
2) Nonstop decay (NSD)
3) No-go decay (NGD)
28
29. How are PTCs distinguished from
the normal termination codon
further downstream?
Effect of tethering a PABP
near a premature
termination codon
29
30. The list of normal NMD substrates:
1) mRNAs with especially long 3′ UTRs
2) mRNAs encoding selenoproteins
3) unknown number of alternatively spliced mRNAs
• NMD may turn out to be an important rapid decay pathway for a variety of
short-lived mRNAs
• Nonstop decay (NSD) targets mRNAs that lack an in-frame termination codon
• Targeting nonstop substrates SKI proteins
30
31. •No-go decay (NGD) targets mRNAs with ribosomes stalled in the
coding region codon
•Targeting of the mRNA involves recruitment of two proteins,
Dom34 and Hbs1, which are homologous to eRF1 and Erf3,
respectively.
•mRNA degradation is initiated by an endonucleolytic cut, and the 5′
and 3′ fragments are digested by the exosome and Xrn1.
31
32. 10. Translationally Silenced mRNAs : RNA Granules
1) RNA granules: translationally silenced mRNA + proteins
2) Germ cell granules & neuronal granules : translational repression
and transport
3) Processing bodies (PBs): mRNA decay components
4) Stress granules (SGs) accumulate in response to stress: Inhibition
of translation
32
33. • The occurrence in germ cells and neurons of macroscopic, cytoplasmic
particles containing mRNA has been known for many years.
• One similarity among all of the known RNA granules is that they harbor
untranslated mRNAs and about 50 to 100 different proteins, depending on
granule type.
•RNA granules = mRNPs + protein
•Germ cell granules (maternal mRNA granules) oocytes
Repression deadenylation
Activation polyadenylation
33
34. • Neuronal granules: similar to maternal mRNA granules
• Processing bodies (PBs): contains proteins involved in mRNA decay
• mRNAs silenced via RNAi and miRNA pathways are present in PBs
• PABPs are not found in PBs, suggesting that deadenylation precedes mRNA
localization into these structures.
• PABPs: dynamic, increasing and decreasing in size and number, and even
disappearing, under different cellular and experimental conditions that affect
translation and decay
• Not all resident mRNAs are doomed for destruction, though; some can be
released for translation 34
35. • Stress granule (SG): only accumulate in response to stress-induced
inhibition of translation initiation
• SGs lack components of the RNA decay machinery, which PBs have, but
include many translational initiation components that PBs lack.
• Both types of particle can coexist in one cell
• the size and numbers of both increase under stress conditions
• mRNAs may be exchanged between the two types of particles
• Granule mRNAs are normally in a dynamic equilibrium with the
population of mRNAs being translated
35
36. 11. Eukaryotic mRNAs Localized to Specific
Regions of a Cell
1)Localization of mRNAs serves diverse functions in single cells and
developing embryos
2)Three mechanisms for the localization of mRNA
3)Localization: cis-elements target mRNA / trans-factors mediate
4)The predominant active transport mechanism involves the directed
movement of mRNPs along cytoskeletal tracks
36
37. • Most mRNAs are probably translated in random locations
• mRNA localization: 3 key functions
1) specific mRNAs in the oocytes
2) asymmetric cell divisions
3) mechanism for the compartmentalization
of the cell intospecialized regions
37
38. • mRNA localization is particularly important for highly polarized cells such
as neurons
• Most mRNAs are translated in the neuron cell body, many mRNAs are
localized to its dendritic and axonal extensions: β-actin mRNA
• Localization of mRNA at neuronal postsynaptic sites seems to be essential
for modifications accompanying learning
• Glial cells myelin sheath
• Plants localize mRNAs to the cortical region of cells and to regions of
polar cell growth
• mRNA localization involves transport from one cell to another. 38
39. • Maternal mRNPs in Drosophila are synthesized and assembled in
surrounding nurse cells and are transferred to the developing oocyte
through cytoplasmic canals
• Plants can export RNAs through plasmodesmata and transport them for
long distances via the phloem vascular system
• 3 mechanisms for the localization of mRNA:
1) The mRNA is uniformly distributed but degraded at all sites except the site of
translation
2) The mRNA is freely diffusible but becomes trapped at the site of translation
3) The mRNA is actively transported to a site where it is translated 39
40. • Active transport is the predominant mechanism for localization:
• All three molecular motor types are exploited: dyneins & kinesins & myosins
4 components:
1) cis-elements on the target mRNA
2) trans-factors that directly or indirectly attach the mRNA to the correct motor protein
3) trans-factors that repress translation
4) an anchoring system at the desired location.
40
41. • Only a few cis-elements, sometimes called zipcodes, have been characterized
• A large number of trans-factors have been associated with localized mRNA
transport and translational repression
• Staufen protein: This multitalented factor has multiple domains that can
couple complexes to both actin- and microtubule-dependent transport pathways
• Cotranscriptional binding of the zipcode element by the protein ZBP1 is
required for localization
• mRNA is committed to localization before it is even processed and exported
from the nucleus
• β-actin mRNA localization is dependent on intact actin fibers in fibroblasts and
intact microtubules in neurons
41
42. Newly exported ASH1 mRNA is attached
to the myosin motor Myo4 via a complex
with the She2 and She3 proteins. The motor
transports the mRNA along actin filaments
to the developing bud
42
43. 5' and 3' modifications controlling RNA
degradation
• RNA degradation is a key process in the regulation of gene expression. In all organisms,
RNA degradation participates in controlling coding and noncoding RNA levels in response
to developmental and environmental cues.
• The constant control of RNA quality prevents potential deleterious effects caused by the
accumulation of aberrant non-coding transcripts or by the translation of defective
messenger RNAs (mRNAs).
• any functional RNA (including a successful pathogenic RNA) must be protected from the
scavenging RNA degradation machinery. Yet, this protection must be temporary, and it
will be overcome at one point because the ultimate fate of any cellular RNA is to be
eliminated. This special issue focuses on modifications deposited at the 5' or the 3'
extremities of RNA, and how these modifications control RNA stability or degradation.
43
44. • Although the decay of eukaryotic RNAs can be initiated by endoribonucleolytic cleavages,
it is mainly carried out by exoribonucleases
• The m7G cap initially is recognized by the nuclear cap binding complex, which facilitates
mRNA export from the nucleus and is later replaced by the essential translation initiation
factor, eIF4E.
• At the other end of the mRNA, the poly(A) tail is bound by poly(A) binding proteins
(PABPs), which facilitate mRNA export from the nucleus and enhance protein synthesis
through interactions with translation initiation factors
• Deadenylated mRNAs can be uridylated or stored in a dormant state to be later re-
adenylated to activate protein synthesis TUTases & non-canonical poly(A)
polymerases
44
45. • Although in bacteria, bulk RNA decay is initiated by endonucleolytic
events, the ends of RNA molecules also play an important role in
controlling RNA fate. Secondary structures may impede the progression
of 3' to 5' exoribonucleases and polyadenylation accelerates the
degradation of RNA decay intermediates facilitating the action of 3' to 5'
exoribonucleases. Moreover, the triphosphate moiety present at the 5'
end of the primary transcripts restricts endonucleolytic cleavage by the
RNaseE and its conversion to monophosphate accelerates the decay rates
of many, but not all, transcripts.
45
46. • Charenton: 5‘ end cap structure Nudix(family hydrolase Dcp2)
• Warkocki: uridylation by TUT7/TUT4 in the cytoplasm induces decay of
various RNA species and protects cells against viruses and LINE-1
retrotransposons
• Zigackova: The key factors involved in the uridylation and degradation of
RNAs, i.e. TUTases and 30 –50 exoribonucleases recognizing uridylated
RNAs.
• Meaux: How uridylation induces decay of replication-dependent histone
mRNAs in mammals? the serine/threonine protein kinase Smg1 & the RNA
helicase Upf1
46
47. The role of nonsense-mediated mRNA decay (NMD)
in regulating gene expression
• NMD results from improper translation termination at stop codons occurring in places of
the mRNP that lack necessary termination stimulating signals and/or contain termination
inhibitors.
• The most well understood is the major cytoplasmic poly(A) binding protein (PABPC1),
which acts as a potent NMD suppressor when bound in the vicinity of the stop codon.
• Slow or stalled translation termination allows the core NMD factor UPF1 – an
ATPdependent RNA helicase – and the SMG1 complex to interact via the release factors
eRF1 and eRF3 with the stalled ribosome. Interaction of UPF2 and possibly UPF3B with
the SMG1- UPF1 complex activates the SMG1 kinase, resulting in the phosphorylation of
several serineglutamine (SQ) and threonine-glutamine TQ motifs in the N and C-terminal
part of UPF1 that flank its helicase domain. 47
48. • The endonuclease SMG6 and the heterodimer SMG5/SMG7 have been found to interact
with phosphorylated UPF1 and to induce the degradation of the targeted mRNA.
• Recent evidence suggests that in human cells NMD is predominantly initiated through a
SMG6-mediated endonucleolytic cleavage near the stop codon and as a backup system
through SMG7-mediated recruitment of the CCR4/NOT complex, which deadenylates the
mRNA, leading to decapping followed by 5’-to-3’ exonucleolytic degradation by XRN1.
• Interestingly, it seems that not all proteins known to be required for NMD in
mammalian cells have homologues in other organisms
• It was discovered early on that the presence of an exon-exon junction located >50
nucleotides downstream of the stop codon was a hallmark of mammalian mRNAs subject
to NMD
48
49. • It was later found that these exon-exon junctions function as enhancers of NMD efficacy
and that long 3’ UTRs lacking exon-exon junctions can also trigger NMD
NMD factors are essential for normal embryonic development and viability
• There is evidence that some NMD factors function in additional cellular pathways apart
from NMD. For example, UPF1 is involved in telomere maintenance, cell cycl progression
and degradation of histone mRNAs and SMG1 and SMG6 were also reported to play a role
in telomere maintenance
• There is evidence suggesting that NMD inactivation is responsible for the observed
developmental defects.
49
50. Tight regulation of NMD activity is required for normal spermatogenesis
• Several lines of evidence support a role for NMD in germ cell development and male
fertility
• Conditional ablation of UPF2 from embryonic Sertoli cells (SC) – somatic cells that nourish
sperm cells through spermatogenesis – in mice leads to testicular atrophy and male sterility
due to depletion of SC and germ cells (GC)
NMD is involved in the integrated stress response
• By downregulating stress-responsive genes in unstressed cells, NMD activity may prevent
the triggering of a stress response upon innocuous stimuli. However, following exposure to
strong stressors, specific mRNAs can evade NMD and are upregulated as part of the stress
response.
50
51. • Autophagy is a surveillance mechanism that rids the cells of cytoplasmic deleterious proteins
and also serves as a source of amino acids during metabolic stress. Many cellular stressors,
like amino acid deprivation and hypoxia, inhibit NMD and activate autophagy, and an
inverse correlation between these two pathways was reported.
• Inhibition of NMD activates autophagy, in a partially ATF4-dependent manner, and increases
intracellular amino acid levels, while NMD hyperactivation blunts stress-induced autophagy
• NMD inhibition by stress-induced eIF2α phosphorylation leads to stabilization of SLC7A11,
increase in intracellular glutathione levels and establishment of an adaptive response to
oxidative stress.
• It was recently reported that NMD inhibition is also part of the pro-apoptotic response to
stress
51
52. RNA viruses are targeted by or able to evade the host NMD machinery
• Since viral genomes present targets for NMD, it is unsurprising that viruses have evolved
mechanisms to protect them from degradation by the host cell NMD machinery. For
example, Hepatitis C Virus (HCV) was shown to inhibit NMD in hepatoma cell lines
NMD factors are autoregulated by the NMD pathway
• Huang: suppression of NMD by NMD factor knockdowns resulted in a significant increase
in NMD factor mRNA levels of UPF1, UPF2, UPF3B, SMG1, SMG5, SMG6 and SMG7.
52
53. NMD factor mutations in human diseases and the therapeutic potential of
modulating NMD activity
• somatic mutations in the UPF1 gene are frequently found in pancreatic adenosquamous
carcinoma (ASC) and in inflammatory myofibroblatic tumors (IMTs)
• In both cases, the mutations reduce UPF1 protein levels and result in impaired NMD in
the tumor tissue relative to adjacent normal tissue
• NMD exerts important biological functions that reach beyond mRNA surveillance.
Although we are still in the beginning of unravelling the different regulatory circuits in
which NMD is implicated, the existing evidence suggests that NMD affects gene
expression in a diverse range of biological contexts. The unifying principle in all these
cases appears to be the occurrence of stop codons in mRNP environments that are not
optimal for fast enough and/or correct translation termination, leading to the degradation
of the respective mRNA. 53
55. 1. Lewin’s-Genes-XII-2018
2. Molecular Biology of the Cell Sixth Edition
3. 5' and 3' modifications controlling RNA degradation: from safeguards to
executioners(2018)
4. Beyond quality control: The role of nonsense-mediated mRNA decay
(NMD) in regulating gene expression(2017)
55