This document provides an overview of RNA-binding proteins (RBPs) and their functions in plants, particularly in response to environmental stresses. It begins with an introduction to RBPs, describing the different types of RNA-binding domains and how RBPs bind to RNA. It then discusses the diverse functions of RBPs in processes like alternative splicing, RNA modification, polyadenylation, and mRNA localization, transport, stability, and translation. A major section explores the role of RBPs in responding to environmental stresses like temperature, osmotic stress, drought, and flooding. It presents examples of specific RBPs that are regulated under stress conditions. The document concludes with discussing future research perspectives on further characterizing plant

1. Beyond transcription: RNA-binding proteins as emerging regulators
of plant response to environmental constraints
Presented by:
Balasaheb Biradar
iabt, UAS Dharwad

2.  Introduction
 Types of Plant RNA-binding proteins (RBPs)
 Diverse functions of RBPs
 RNA-binding proteins in response to environmental stresses
 Future perspectives
 Conclusion
OUTLINE OF SEMINAR

3. 3
INTRODUCION
RNA-binding proteins (RBPs) that bind to RNA recognition motifs (RRMs) of
double or single stranded RNA in cells and form ribonucleoprotein (RNP) complexes.
RBPs in nucleus exist as complexes of protein and pre-mRNA called heterogeneous
ribonucleoprotein (hnRNPs).
Eukaryotic cells to utilize RNA exons in various arrangements, giving rise to a unique
RNP (ribonucleoprotein) for each RNA.
RNA-binding proteins (RBPs) govern many aspects of RNA metabolism Pre-mRNA
processing, transport, stability/decay and translation.
Arabidopsis thaliana genome contains 200 putative RBP genes while 250 RBP genes in
Oryza sativa.
Eukaryotic genomes encode a large number of RBPs yeast 5–8% and in Caenorhabditis
elegans and Drosophila melanogaster, approximately 2%.
Why do eukaryotes need so many – hundreds and perhaps thousands of – RBPs?

4. RNA-binding proteins (RBPs) are numerous and diverse
Some well-characterized RNA-binding domains :-
• RNA-binding domain (RBD), also known as RNP and RRM domain
• K-homology (KH) domain (type I and type II)
• RGG (Arg-Gly-Gly) box
• DEAD/DEAH box
• Zinc finger (ZnF, mostly C-x8-X-x5-X-x3-H)
• Double stranded RNA-binding domain (dsRBD)
• Pumilio / FBF (PUF or Pum-HD) domain
• Piwi/Argonaute/Zwille (PAZ) domain

5. Fig. RNA-binding domains of RBPs
Alfredo et al., 2012

6. Fig. How RNA-binding modules recognize RNA

7. Fig. Human Pumilio protein Fig. RRM from yeast Hrp1 Fig. KH domains of NusA
Fig. Two dsRBDs of ADAR2 Fig. Polypyrimidine-tract binding
(PTB) protein
Fig. TFIIIA–RNA complex
RNA-binding modules function together to recognize a specific RNA

8. Table:- Common RNA-binding domains and their properties

9. Fig. RNA-binding modules are combined to perform multiple functional roles
Fig. Modular architecture allows for the regulation of the catalytic activity of enzymatic domains.

10. Fig. RNA-binding proteins (RBPs) function in multiple cellular processes
Tinaet al., 2008

11. Table :- Physiological roles of RNA-binding proteins
Gideon et al., 2007

12.  Methods to identify and characterize the RBPs and the RNAs with
which they interact.
 RIP assay :- which combines reversible cross-linking with formaldehyde
followed by immunoprecipitation and RT-PCR.
 Cross-linking and immunoprecipitation ( CLIP):- Recently, this method adapted,
using tagged proteins and including an immunoprecipitation step following
cross-linking.
 A yeast-three hybrid system:- Has been devised as a screening method to
identify RBPs and their target RNAs

13. Fig. Reversible cross-linking combined with immunoprecipitation (RIP)

14. Fig. A yeast-three hybrid systemFig. Cross-linking and immunoprecipitation (CLIP)

15.  Ultraviolet (UV) cross-linking:- (in vivo)
• The hnRNP and messenger RNP (mRNP) complexes were initially isolated by
ultraviolet (UV) cross-linking of RNA–protein complexes in vivo
• It is a reliable and effective method used to detect RNA–protein interactions,
and association of proteins with RNAs that could occur after cell lysis
 An affinity tag :- Facilitate the isolation of an RBP of interest, followed by
analysis of associated RNAs using microarrays,
 Bioinformatics approaches :- To identify RNA targets if a consensus and non-
degenerate RNA-binding sequence is known

16. Functions of RBPs
 Alternative splicing
 RNA modification
 Polyadenylation
 mRNA export
 mRNA localization
 Translation
 mRNA turnover
 Multi-functional proteins

17.  Alternative splicing :-
• Mechanism by which different forms of mature mRNAs generated from same
gene.
• Variations in the incorporation of the exons into mRNA leads to the production
of more than one related protein.
• Ex. Neuronal specific RNA-binding proteins (NOVA), Defect cause
Paraneoplastic neurological disorder (PND).
 RNA modification :-
• Change in nucleotide content of RNA and extends the diversity of gene
products.
• Conversion of adenosine to inosine in an enzymatic reaction catalyzed
by ADAR.
• Ex. Glutamine is converted to arginine.

18. Polyadenylation :-
• Addition of a “tail” of adenylate residues to an RNA transcript about 20 bases
downstream of AAUAAA sequence within 3’ untranslated region.
• Polyadenylation of mRNA has strong effect on its nuclear transport, translation
efficiency, and stability.
• Ex. CPSF (Cleavage and polyadenylation specificity factor) with PABP activate
poly A polymerase.
 mRNA export :-
• After processing is complete, mRNA needs to be transported from the cell
nucleus to cytoplasm. Ex. TAP/NXF1
1) Generation of a cargo-carrier complex in the nucleus
2) Translocation of the complex through the nuclear pore complex
3) Release of the cargo into cytoplasm.

19.  mRNA localization :-
 Localization of transcript to specific region of cell during development.
 Through mRNA localization proteins are transcribed in their intended target site of the
cell.
 ZBP1 (Zipcode binding protein) binds to beta-actin mRNA at the site of 3’ UTR of β-actin
mRNA and moves mRNA into the cytoplasm.
 It then localizes this mRNA to the lamella region of several asymmetric cell types where
it can then be translated.
 Translation :-
 Translational regulation provides rapid mechanism to control gene expression.
 Rather than controlling gene expression at transcriptional level, mRNA is already
transcribed but recruitment of ribosomes is controlled.
 ZBP1 its role in localization of B-actin mRNA is also involved in the translational
repression of beta-actin mRNA by blocking translation initiation.

20.  mRNA turnover :-
• Translation is tightly coupled to mRNA turnover and regulated mRNA
stability.
• ELAV/Hu proteins are involved in the stability and translation of early
response gene and AU-rich transcripts predominantly in neurons.
 Multi-functional proteins :-
• Many RBPs, ex. hnRNP and serine/arginine-rich (SR) proteins, bind to
multiple sites on numerous RNAs to function in diverse processes.
• hnRNP A1 protein bind to exonic splicing silencer sequences and regulate
alternative splicing by antagonizing the SR splicing factors.
• hnRNP A1 has been shown to stimulate telomerase activity by associating
with telomere ends.

21. Fig. The function of RBPs in the regulation of post-transcriptional gene expression
Tina et al ., 2008

22. Fig. The RBPs in function of gene regulation and post-transcriptional gene expression
Zdravko et al., 2009

23. RNA-binding proteins in response to environmental stresses
• An alternative mechanism to rapidly reprogram the plant’s transcriptome in response to
environmental stresses is via transcript stability, degradation and turnover
• RBPs are emerging as crucial group of proteins involved posttranscriptional changes
triggered in plants in response to variable external conditions
• RBPs expression and/or activity were found to be regulated in response to environmental
variables
 Temperature stress
 Osmotic stress
 RBPs and ABA-mediated stress response
 Flooding stress
 Draught stress

24. Alfedo et al., 2012

26.  Material and methods :-
 Growth and stress conditions
 cDNA library construction, screening and molecular cloning
 In silico analysis of rice genome for OsGR-RBP4 homologues
 Southern and Northern analysis
 protein isolation and Western blotting
Yeast thermo tolerance assays
Polysomal fraction analysis
Immunolocalization of OsGR-RBP4

27. (A) Phylogenetic relationship of rice GR-RBPs
determined by CLUSTAL-V of DNAStar
(B) Schematic model of the OsGR-RBP4
protein
Fig. Hydropathy plot of OsGR-RBP4
Results:-
N C
Hydrophobic

28. (C) Alignment report of homologous GR-RBPs reported from different plant and animal systems

29. Fig. Southern blot analysis of Osgr-rbp4
gene.
Fig. Expression profiles of Osgr-rbp4 at different time points

30. Fig. Expression profiles of Osgr-rbp4 in response to different stresses
200Mm
100Mm

31. Fig. Soluble proteins isolation from unstressed
and HS rice tissues
Fig. Expression levels of OsGR-RBP4 during recovery

32. Fig. Thermo tolerance assays using yeast cells expressing OsGR-RBP4
Fig. Analysis of OsGR-RBP4 in rice polysomal fraction

33. Fig. Localization of OsGR-RBP4 in rice leaf sections

35. Material and methods :-
 Plant materials and growth conditions
 High light and anoxia treatments for DD-PCR and semi-quantitative RT-PCR
experiments
 Isolation of OsDEGs with Gene Fishing DEG kit and Semi-quantitative reverse
transcription-PCR
 Drought, salt, ABA, MV and cold treatments and semi-quantitative RT-PCR
experiments
 Plasmid construction and rice transformation
 Measurement of chlorophyll fluorescence
 Extraction of xanthophyll pigments and HPLC assay

36. Fig. Expression patterns of the selected twenty-two
OsDEGs under high light and anoxia treatments
Fig. The nine OsDEGs had the same expression
patterns as those in the DD-PCR results

37. Fig. RT-PCR analysis at 0, 1, 2, 3 and 4 h
after highlight treatment
Fig. RT-PCR analysis at 0, 1, 2, 3 and
4 h after anoxia treatment
Fig. RT-PCR analysis at 0, 24, 48 and
72 h after drought treatment
Fig. RT-PCR analysis at 0, 200, 300 and
400 mM NaCl treatments
Fig. RT-PCR analysis at 0, 100, 200
and 300 lM ABA treatments
Fig. RT-PCR analysis at 0, 10, 20 and
30 lM MV treatments

38. 38
Fig. OsDEG10 amino acid sequence
Fig. Multiple alignments of RRMs of OsDEG10, two rice OsDEG10 homologs, Os03g17060 and Os03g36490, and three
Arabidopsis small RBPs
Fig. RT-PCR analysis of two rice OsDEG10 homologs, Os03g17060 and Os03g36490, after high light, NaCl, MV and
cold treatments

39. Fig. Schematic map of OsDEG10 RNAi binary vector
Fig. Semi-quantitative RT-PCR analysis of OsDEG10 transcript in 2-week-old wild-type plants and OsDEG10
RNAi T1 transgenic plants
Fig.Changes in PS II activity (Fv/Fm) of wild-
type and OsDEG10 RNAi T2 plant
Fig. The contents of xanthophyll pigments in
wild-type and OsDEG10 RNAi T2 plants
Fig. Changes in PS II activity (Fv/Fm) of wild-
type and OsDEG10 RNAi T2 plants

40. 40

41. MATERIALS AND METHODS :-
Plant material and stress treatments
cDNA cloning for a GRP
RNA blot hybridization
Production and purification of recombinant NtGRP1 proteins from E.coli
Antibody production and protein blot analysis
Binding assay on DNA and RNA
Coupled In vitro transcription/translation
Oligonucleotide competition assay

42. Fig. Nucleotide and deduced amino acid
sequences of NtGRP1
Fig. Multiple alignment of amino acid sequences of NtGRP1
and other plant GRPs in plants

43. Fig. DNA blot hybridization analysis of
tobacco genome for NtGRP1
Fig. Effects of ABA and various stresses on the level of
NtGRP1 transcript

44. Fig. H6NtGRP1 overexpressed in
E.coli was purified
Fig. North wester blot analysis of
NtGRP1
Fig. Schematic representation of NtPGR1 and its
truncated form
Fig. Northwestern blot analysis for H6NtGRP1,
H6NtGRP1 and H6NtGR1
Fig. Analysis of mRNA binding activity of
NtGRP1 by gel mobility shift assay

45. 45
Fig. Analysis of DNA binding activity of NtGRP1 by gel mobility shift assay

46. Fig. Inhibitory activity of NtGRP1 on the expression of luciferase gene H6NtGRP1 (25-300 μM) was added
to the in vitro coupled transcription/translation system of pT7-luciferase
Fig. Suppression activity of NtGRP1 on the expression of luciferase gene confirmed by luminescence of
luciferase reaction mixture

47. Fig. In vitro translation of luciferase mRNA was carried out in the presence of H6NtGRP1 and oligonucleotides DNA
Fig. In vitro translation of luciferase mRNA was carried out in the presence of H6NtGRP1 and oligonucleotides RNA

49.  Materials and Methods :-
• Plant Materials and Drought Treatment
• Total RNA Extraction and mRNA Isolation
• Suppression Subtractive Hybridization (SSH) cDNA Library Construction and
EST Analysis
• cDNA Synthesis and In Silico Cloning of MpGR-RBP1
• Analyses of Sequences and Phylogenetics
• Quantitative Real-Time PCR

50. Fig. Nucleotide sequence of MpGR-RBP1 cDNA and deduced amino acid sequence

51. Table :- Comparison of the deduced amino acid sequences from MpGR-RBP1 with known
related protein sequences

52. Fig. (A) Putative conserved domain structure and schematic model for predicted amino acid sequence shows amino-
terminal RRM and carboxyl-terminal glycine-rich domain. (B) Putative conserved domain structure of RRM
Fig. Phylogenetic tree of MpGR-RBP1 with other plant GR-RBPs

53. Fig. Quantitative real-time RT-PCR analysis of the MpGR-RBP1 expression level in leaves and roots from
Malus prunifolia under drought stress

54. Fig. Semiquantitative RT-PCR analysis of MpGR-RBP1 expression under well-watered conditions (CK) and in
response to drought (4, 6, 8, 10, or 12 days)

55. FUTURE PERSPECTIVES
 Experimental characterization of genes encoding putative RBPs in plant genomes.
 Plant-specific RBP genes, their role in stability, translation and their involvement in
plant’s stress response.
 Systematic identification of target mRNAs for individual plant RBPs.
 Contribution of RBPs in the silencing machinery through small interfering RNAs (siRNA)
 Potential role of RBPs in the stress-induced plant developmental changes.
 Need more information about biochemical, genetic as well as bioinformatics analysis of
RBPs about which little is known.

56. Conclusion

57. THANK YOU..