This document discusses non-coding RNA and its important regulatory roles. It notes that while RNA was originally thought to only act as a messenger between DNA and protein, recent evidence shows extensive transcription of non-coding sequences and diverse biological functions of non-coding RNA beyond information transfer. Different types of non-coding RNA are involved in fundamental cellular processes as well as higher-level regulatory functions. Studies have found thousands of non-coding transcripts expressed in complex organisms, and non-coding RNA is emerging as a key player in gene regulation.

1. Non-coding RNA: A Review
(Unveiling the Hidden World of Gene Regulation)
John S. Mattick and Igor V. Makunin
Presented By :
Babita Neupane
Date: 30th Aug, 2023
Mattick, J., Makunin, I., & Non-coding, R. (2006). Human moleculargenetics 15 Spec No 1. R17.

2. About the Journal
✓Human Molecular Genetics under Oxford Academic
(Oxford University Press)- >500 Journals
✓Publishes high quality research articles under human
molecular genetic disease mechanisms from the analysis of
mutated genes and disease susceptibility through to
therapeutics.
✓Publishes one or two review issues yearly.
✓Impact factor = 3.5 (2022)
✓Total cites =41216
✓Biochemistry & Molecular Biology= 150/285
✓Genetics and Heredity =65/171
✓Contribution :This paper, "Non-coding RNA" published in
this journal in Volume 15, Review issue I, pages 17-29, is
authored by John S. Mattick and Igor V. Makunin.
✓Most cited paper yet from this jornal(1.9K citation).

3. About the author
Professor John S. Mattick
Science
School of Biotech& Biomolecular Science
• SHARP Professor of RNA Biology at UNSW
Sydney.
• Executive of Genomics England (2018-2019),
• Director of the Garvan Institute of Medical Research
in Sydney (2012-2018)
• B.Sc. (First Class Honours, Biochemistry) 1972,
University of Sydney
• Ph.D. in Biochemistry 1978, Monash University,
Melbourne.
• Published over 300 research articles and reviews,
which have been cited over 80,000times
Igor V. Makunin, Phd.
Senior Bioinformatician
QCIF
• Ph.D. in Biology(1996).
• Currently involved in Queensland Cyber
Infrastructure Foundation (QCIF).
• Trained as geneticist and molecular biologist.
• Later switched to comparative genomics and
non-codingRNA.
• Migrate from wet lab to bioinformatics and
analysis of high throughputsequencing.
• Research Interest : Comparative Genomics and
Analysis of High throughput sequencing..

4. Introduction
✓Historically, ncRNAs like rRNAs, tRNAs, snRNAs, and
snoRNAs were thought to have generic cellular functions.
✓Molecular biology's core principle proposed RNA's role as an
intermediary between DNA and protein.
✓Protein-centric view assumed most genetic information is
expressed as proteins, with RNA mainly acting as a messenger.
✓Non-coding sequences in eukaryotes often considered
evolutionarydebris.
✓However, recent insights show extensive transcription of those
inert sequences.
✓RNA demonstrates diverse biological functions beyond
information transfer.
✓RNA, including ncRNAs, emerges as a key player in gene
regulation.

5. Objectives
❑Study DiverseFunctionsof ncRNAs
❑Study role of ncRNAin widespreadtranscription
mechanism
❑Understandfunctional complexityof ncRNAs.
❑Study impact of ncRNAs on characteristicsand diseases.
❑Understandfuture prospectsof study of ncRNAs.

6. Expansion of ncRNA’s and RNA metabolism in
Eukaryotes
✓Prokaryotes have limited small ncRNAs regulating translation/stability
whereas Eukaryotes have abundance of diverse small ncRNAs, co-
expressed with mRNAs released by cleavage after transcription.
✓Archaea and bacteria possess homologs of Argonaute, a family of RNA-
binding endonucleases central to the action of micro- RNAs (miRNAs)
and small interfering RNAs (siRNAs) in Eukaryotes.
✓Prokaryotes genome is dominated by protein-coding sequences (80–
95%) whereas eukaryotes genome dominated by ncRNA .
✓Eukaryotes have more developed RNA processing and signaling systems
linked to sophisticated pathways of gene regulation and complex genetic
phenomena.
✓Large repertoire of RNA-binding proteins, contributing to regulatory
complexity.
✓Eukaryotes utilize RNA as a digital regulatory solution, impacting
developmental programming and species differences.

7. 1. Transcriptional regulation
✓ U1 snRNA regulates binding of RNA Polymerase II with TFIIH to initiate
transcription process.
✓ Also interacts with cyclin H indicating involvement in cell regulation
2. ProgrammedCell Death
✓ Small conserved nuclear RNA 7SK RNA acts with HEXIM1 &2 proteins ; depletion
of this the 7SK shows apoptosis in HeLa cells .
3. Chromatin architecture and Epigenetic memory
✓ Small ncRNA similar to H/ACA snoRNA is component of telomerase; mutation in this
ncRNA results in dominant dyskeratosis congenita.
✓ In human-chicken hybrid cell; mutation in dicer(key component of siRNA/miRNA )
results premature separation of sister chromatids and cell death.
4. Cell biological process
✓ ncRNA 7SL;regulates protein localization; component of SRP; identify signal peptides
of protein)
✓ 13MDA vault complex is huge ribonucleoprotein complex present in cytoplasm
presumed to help in cellular transport.
✓ Vault complex hvg-1 and hvg-2, have a binding affinity for mitoxantrone, a
chemotherapy drug utilized in breast cancer, myeloid leukemia, and non-Hodgkin's
lymphoma treatment.
INFRASTRUCTURAL
ncRNAs
❑ Involved in fundamental cellular
processes like protein synthesis,
RNA splicing & catalytic
activity itself but also found to
have regulatory functions.
❑ Include tRNAs, rRNAs,
spliceosomal uRNAs or
‘snRNAs’ and the common
‘snoRNAs .

8. Cis-Acting regulatory sequences in
Non-Coding regions of mRNAs and
pre-mRNAs
✓Regulatory RNAs base-pair with complementary sequences, forming complexes recognized by generic
infrastructure.
✓UTRs in mRNAs receive trans-acting signals via regulatory RNA sequences, influencing stability, translation,
and localization.
✓Riboswitches bind metabolites, controllingmRNA translation/stability; found in bacteria and eukaryotes.
✓miRNAs influence evolution and regulate mRNA by base sequence recognition.
✓UTR length reflects mRNA regulation sophistication;longer in complex organisms.
✓Cis-acting sequences near splice junctionsinfluence alternative splice site selection.
✓Nucleotide conservation higher around alternative splice sites; complex RNA:RNA interactions affect splicing
control.
✓Some protein-codingsequenceshave dual roles, targeted by regulatory miRNAs and siRNAs.
✓Ultra-conserved sequences at splice sites; proteins may have multifunctionalroles.
✓RNAs combine digital and analog functions; deeper understandingneeded for complex networks.

9. Large numbers of ncRNAs expressed from the mammalian genome
✓ Only about 1.2% of the genome encodes protein-coding
genes, yet as much as 60-70% is transcribed, with both
strands involved. Current estimates are conservative,
considering unexplored cellular contexts.
✓ Mouse genome studies reveal numerous non-coding
transcripts (over 34,000) not encoding proteins,
suggesting vast transcriptional complexity beyond
expectations. Transcriptional depth remains under-
explored, impacting mRNA regulation understanding.
✓ Around 22% of human transcription clusters form sense-
antisense pairs, with considerable evolutionary
conservation. Over 70% of mouse transcriptional units
overlap with opposite-strand transcripts. Sense-antisense
interactions hint at greater transcription extent than
genome size.
✓ Tiling array and MPSS studies identify thousands of non-
coding transcripts in intergenic and intronic regions,
significantly expanding known transcriptomes. Secondary
structure analyses predict functional ncRNAs.
✓ Genome tiling array studies suggest extensive
transcription of non-repetitive sequences in cell-specific
manners, with overlaps between opposite and same
strands, challenging conventional gene and exon
definitions.
✓ RACE analysis confirms intricate networks of
overlapping transcripts and unrecognized exons,
complicating genotype-phenotype correlations and gene
definitions, shaping the view of genes as complex
transcription clusters.
✓ A significant proportion of transcripts in human and
mouse lies within unstudied nuclear polyA+ and polyA−
fractions, revealing that the full extent of the
transcriptome complexity remains largely uncharted.

10. Small Regulatory ncRNAs: snoRNAs (Small Nuclear RNA)
What are SnoRNAs?
✓They are compact RNA molecules, usually
consisting of 60 to 300 building blocks.
✓They serve as guides, pairing with target
RNAs and directing specific chemical
changes.
✓There are two primary types: box C/D
snoRNAs for 2′-O-ribose-methylation and
box H/ACA snoRNAs for
pseudouridylation.
✓Initially thought to modify only ribosomal
RNAs (rRNAs) during ribosome
construction, snoRNAs have since shown
their versatility by influencing various
RNAs, including messenger RNAs
(mRNAs) and small nuclear RNAs
(snRNAs).
How they origin and where they
located ?
✓ Many snoRNAs in mammals are
derived from introns, sections within
genes.
✓ Some human C/D snoRNAs are
independently transcribed,
distinguished by special caps at their
beginning.
✓ A subgroup of H/ACA snoRNAs
resides in small nuclear
compartments known as Cajal
bodies, often called scaRNAs.
✓ Telomerase RNA, which helps
maintain chromosome ends, also
makes appearances in Cajal bodies.
Regulation & Diversity of snoRNAs
✓They have specific roles in certain
tissues or developmental stages,
showcasing their regulatory
functions.
✓ There are also "orphan" snoRNAs
whose purposes remain a mystery.
✓One snoRNA, found in Prader-Willi
syndrome patients, disrupts normal
gene splicing.
✓However, snoRNAs are just the tip
of the iceberg; many more are yet to
be discovered, and this likely
extends to other uncharacterized
small regulatory RNAs waiting to be
unveiled.

11. Small Regulatory ncRNAs: miRNAs/siRNA
✓ MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are tiny RNA molecules, about 22 units long, derived
from hairpin or double-stranded RNA.
✓ miRNAs can hinder translation via partial matching with target mRNAs, whereas siRNAs can destroy target RNAs
when there's a perfect match, termed RNAi.
✓ About one-third of human protein-coding genes are controlled by miRNAs. siRNAs from repeats help shape silenced
chromatin in chromosome dynamics, especially in yeast.
✓ miRNAs stem from introns and exons of coding and non-coding transcripts produced by RNA polymerase II.
✓ They play central roles in various developmental processes, cell activities, and brain functions.
✓ miRNA dysregulation is tied to diseases, including cancer; miRNA profiling can aid cancer diagnosis.
✓ Some miRNAs act like proto-oncogenes, encouraging tumor growth.
✓ miRNA targets aren't limited to mRNA 3'-UTRs; they can appear anywhere in functional transcripts.
✓ Hundreds of miRNAs are confirmed experimentally, many more predicted computationally.
✓ Known miRNAs are highly conserved, but some evolve quickly, co-varying with targets.
✓ Growing research unveils many new human miRNAs, some species-specific; more miRNAs likely exist.

12. Biological Roles of ncRNA
✓Non-coding RNAs (ncRNAs) have a diverse array of functions, spanning chromosome dynamics,
splicing, RNA editing, translational control, and mRNA degradation. Their reach into gene
regulation across eukaryotes is vast.
✓RNA's involvement in chromatin remodeling and epigenetic memory hints at RNA signaling,
although mechanisms remain uncertain. Transcription's role as a regulator itself or as a product of
transcription interference is debated.
✓Emerging evidence points to ncRNA-guided splicing regulation, influencing splice site selection
and accessibility to splicing machinery.
✓Transcription appears regulated by ncRNAs; RNA polymerase II activity is influenced by ncRNA
signaling. Certain transcription factors and chromatin-modifying proteins exhibit RNA affinity.
✓ncRNAs like steroid receptor RNA activator (SRA) impact hormone receptor activity. Stress
responses involve ncRNAs; B2 and BC200 play roles in heat shock responses.
✓ncRNAs scaffold complex assembly, such as rRNA in ribosomes and 7SL RNA in the SRP
complex. NRON serves as a scaffold for nuclear transcription factor trafficking.
✓ The multifaceted roles of ncRNAs highlight their central place in gene regulation and complex
cellular processes.

13. INTRONS AS SOURCE OF FUNCTIONAL ncRNA
✓Introns might be potential source of regulatory ncRNAs ; operates in tandem with
protein-coding sequences to transmit regulatory cues to other genes & transcripts.
✓Almost all snoRNA & large portion of miRNA in animals encoded from introns.
✓Introns may be processed to smaller RNAs; rather than being discarded after
transcription.
✓In an experiment performed in HeLa cell introducing different intronic sequence in
specific genes showed variations in their activity.
✓Complex organism have more introns, enhancing complexity in gene regulation and
expression.
✓They collaborate with protein-coding parts of genes to send regulatory messages,
and recent discoveries highlight their involvement in gene regulation, contributing to
the complexity of our genetic makeup.

14. Future Avenues for ncRNA Research
✓Expanding Functions
Discover diverse roles of ncRNAs beyond tradition.
✓Therapeutic Potential
Develop ncRNA-based therapies and interventions.
✓Evolutionary Implications
Explore ncRNAs' role in genetic diversity and evolution.
✓Advanced Technologies
Utilize CRISPR and advanced sequencing for precise manipulation.
✓Clinical Applications
Create ncRNA-based diagnostics and disease treatments.

15. CONCLUSION
✓Genetic programming in complex organisms involves an extensive array of non-coding RNAs
(ncRNAs), forming hidden molecular genetic signals that regulate developmental pathways.
✓The abundance of ncRNAs in cells, though diverse in function, mainly contributes to directing
intricate developmental processes, critical in highly structured organisms like humans.
✓The coexistence of a sophisticated RNA-based regulatory system alongside common proteomes
explains the vast diversity observed in complex organisms, resolving the paradox of their distinct
traits.
✓These ncRNAs remained concealed due to their sheer numbers and complex population
characteristics, detected only with advanced methods like sensitive genetic screens, genome
sequencing, and bioinformatics.
✓Rapid evolution of functional ncRNAs challenges the notion of non-functionality; they might instead
adapt easily due to different constraints and positive selection linked to phenotypic variation.
✓Extensive conservation of non-coding sequences, possibly up to 10% of the genome, implies
functionality and challenges traditional gene regulation concepts, while bioinformatics and large-scale
sequencing promise insights into their roles and therapeutic potential.

16. THANK YOU !