5' cap

Its about RNA 5' capping and its exciting applications..

1. 5’terminal m7G cap of messenger RNA: structure and function By Sukanya Varape Roll No. 1385

2. Why the cap was evolved only in Eukaryotes? Loss of Shine- Dulgarno base pairing in eukaryotes which is a means of directing the ribosome to the mRNA Eukaryote use ‘cap’ as an alternative signal for directing translational machinery to the mRNA Appearance of 5’exorionuclease only in eukaryotes Existence of capping enzymes only in eukaryotes Specificity of Guanylytransferase

3. Structure of 7- methyl guanosine cap 5’ to 5’ triphosphate bridge( phosphoanhydride bonds) Methyl group on N-7 position

4. Co-transcriptional capping Carboxy -terminal domain ~30 nucleotide Addition of guanine in opposite direction

5. Structure and mechanism of the RNA triphosphatase enzymes Mechanically and structurally they are diverse Two families The divalent- cation -dependent RNA triphosphatase - Found in protozoa, eukaryotic viruses and fungi The metal-independent class of triphosphatase enzyme- Found in metazoa and plants

6. Structure and mechanism of the RNA guanylyltransferase enzymes Lysine side chain It have two structural domains N-terminal nucleotidyl transferae C-terminal OB fold domain It resembles DNA and RNA ligases

7. Structure and mechanism of the mRNA cap (guanine-N7) methyltransferase It’s structure has revealed that, enzyme contains separate ligand - binding pocket for AdoMet (methyl donor) and cap guanine(methyl acceptor)

8. Types of caps

9. The exception RNA dependent RNA polymerase of Vesicular Stomatitis Virus(VSV) incorporates GDP into the cap structure

10. Functions of m7G cap Essential for the growth of eukaryotic cells Marks the translational start site Protects mRNA from degradation by 5’exoribonucleases Methyl group facilitate translation Participate in mRNA splicing and enhances its accuracy Binds to nuclear cap binding complex to facilitate mRNA transport to cytoplasm Specificity of Gunylytransferase ensures that the caps are added only to the intact transcripts and not to endolyticaly cleaved once.

11. Up regulators: 1.Myc 2.E2F1-cell proliferation transcription factor 3.Importin alpha Down regulators: 1.Cdc34-ubiquitin-conjugating enzyme 2.SAHH-S-adenosylhomocystine hydrolyase

12. 1.Specificity 2.Potency

13. mammalian cells contain a surveillance machinery anchored by the DXO and Xrn2 factors that will rapidly degrade incorrectly capped pre-mRNAs Possible Target proteins: Viral TAT protein Mammalian PMIT enzyme

14. mRNAs with short poly-A tails are captured poorly by oligo-dT based methods Recombinant human cap-binding protein eIF4E was used to capture mRNAs on the basis of the 5’ cap structure With mRNA capture by the 5’ 7-methyl guanosine cap provides a more complete view of the transcriptome and can be used to assay changes in mRNA poly-A tail length on a genome-wide scale.

15. Based preparation and sequencing of concatamers of DNA tags deriving from the initial 20 nucleotides from 5’ end mRNAs CAGE Allows high-throughout gene expression analysis and the profiling of transcriptional start points (TSP), including promoter usage Analysis

16. References Amiya k. Banerjee , 5 '-Terminal Cap Structure in Eukaryotic Messenger Ribonucleic Acids, MICROBIOLOGICAL REVIEWS , vol.2 June 1980 Stewart Shuman, What messenger RNA Capping tells us about the eukaryotic evolution, Nature reviews, MolCellBio , Vol. 3 Aug. 2002 Meigang Gu and Christopher D Lima, Processing the message: structural insights into capping and decapping mRNA, Current Opinion in Structural Biology, 15:99–106, 2005 Victoria H. COWLING, Regulation of mRNA cap methylation , Biochem. J. (2010) 425, 295–302 Tomoaki Ogino and Amiya K. Banerjee Unconventional Mechanism of mRNACapping by the RNA-Dependent RNA Polymerase of Vesicular Stomatitis Virus Molecular Cell 25, 85–97, January 12, 2007 Jeffrey Wilusz , Putting an ‘End’ to HIV mRNAs: capping and polyadenylation as potential therapeutic targets, Wilusz AIDS Research and Therapy 2013, 10:31 François Ferron , Etienne Decroly , Barbara Selisko , Bruno Canard, The viral RNA capping machinery as a target for antiviral drugs, Antiviral Research 96 (2012) 21–31 Michael D. Blower et al, Combining Different mRNA Capture Methods to Analyze the Transcriptome : Analysis of the Xenopus laevis Transcriptome , PLOS ONE ,October 2013, Volume 8, Issue 10 Toshiyuki Shiraki , Cap analysis gene expression for high-throughput analysis of transcriptional starting point and identification of promoter usage, PNAS December 23, 2003 vol. 100 no. 26

17. Anne-Claude Gingras , Nature Reviews Molecular Cell Biology AOP , published online 14 october 2009

18. Hats Off to you Cap!!! Thank you