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Eric Martinez Japinder Grewal Bio Sci D145 February 13, 2014

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Eric Martinez Japinder Grewal Bio Sci D145 February 13, 2014

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  1. RNA Maps Reveal New RNA Classes and a Possible Function for Pervasive TranscriptionPhilipp Kapranov, Jill Cheng, SujitDike, David A. Nix, Radharani Duttagupta,1Aarron T. Willingham, Peter F. Stadler, Jana Hertel, JörgHackermüller, Ivo L. Hofacker,Ian Bell, Evelyn Cheung, JorgDrenkow, Erica Dumais, Sandeep Patel, Gregg Helt,Madhavan Ganesh, Srinka Ghosh, Antonio Piccolboni, Victor Sementchenko, HariTammana, Thomas R. Gingeras Eric Martinez Japinder Grewal Bio Sci D145 February 13, 2014

  2. Background:The Central Dogma Of Biology: Not Always True? • Seeing RNAs mapping out specific functions • Not going the usual route of DNA – RNA – Protein • 10X noncoding sequence outnumbers coding sequence • Lots of RNA (not all coding)

  3. Relevance of the Study • Pervasive transcription: large percentage of DNA in mammalian genomics is transcribed • 93% of human genome transcribed in one tissue • Suggests genome consists of functional DNA • Even long noncoding strands • Lots of RNA that is unmarked and reduced protein coding ability • So what does it do?

  4. Outline of the Study • What was Studied: • A genome wide study of origins and associations of human nuclear and cystolic RNAs -long (lRNAs) and short (sRNAs) • How: • Mapped detected RNAs with markers and assessed the potential to process short RNAs (sRNAs) • It Suggests: • Unnannotated RNAs have a major role in transcribing sRNAs (primary transcripts) • Syntenically conserved with weaving pattern of human transcriptome

  5. Materials and Techniques • 8 Cell Lines: • lRNA from sub-cellular cytosolic compartment • sRNA( nuclear Pol(A)+) obtained from cancer cell lines • HeLa - derived from cervical cancer cell - Highly prolific and durable • Hep G2- Human liver carcinoma cell line

  6. Marterials and Techniques… • Tiling array (micro array)- mapped the complex transcribed regions of the human genome and used to compare with previous studies. • Two formats from results- Graphs and Transfrags • Used to create a genealogy

  7. Materials and Techniques… • Showed relation of nucleotide bases and the transfrags from both lRNAs and sRNAs

  8. Materials and Techniques… • lRNA similar to previous studies- cell lines produced similar unannotated and unannotated transcription patterns. • 1.1% covered by Transcribed Fragments- Represents sRNA • Northern Blot- size estimates for sRNA • qRT- PCR- Presence and abundance of sRNA • phastCons scores used to discard Transfrags that didn’t have coverage.

  9. 3 Classes of sRNA (TASRs, PASRs, and long transcripts(PALRS)) • PASRs and TASRs characteristics support biological significance of sRNA • Northern Hybridization analysis- used to find PASRs abd TASRs vary in length • 5’-3’ RACE analysis done for lRNAs(PALRS)

  10. Mouse vs. Humans • Mouse STO and R1 and human HepG2 and HeLacell lines used to make sRNA maps in syntenic regions

  11. Pros and Cons of Genomic Tiling Microarrays • Pros • Investigate protein binding • Allows for analyzing of the noncoding regions • Maps RNAs and not just coding sequences (transcriptome) • Cons • Expensive • No clearly defined start or stop regions of interest identified • "transcriptional noise" produced by its ultra-sensitive detection capability • give only chromosome and position numbers (more sequencing needed)

  12. Importance (Big Picture) • Map a “genealogy” of origins of RNA (4th and 5th paragraph) • Coding regions much more conserved (why are noncoding regions being conserved?) • lRNAtransfrags can represent nuclear primary transcripts encoding conserved functional sRNAs (precursors)

  13. Other Important Findings • Large fraction of protein coding genes expressed only in 1st exon and intron • Transcription based on length of transcripts made from start site of gene locus • Predict conservation of transcripts from transcribed region • nucleus, cytosol, or processed sRNAs • PASRs (promoter associated short RNAs) align with boundaries of PALRs • Genomic loci conserved (common processing signals) • Ends of half human protein-coding genes have PASRs or TASRs (termini associated short RNAs) • Large regions in seq. have sRNAs (chromatin alternation fxn)

  14. Further Study: Possible functions and Implications • Multiple transcripts at 5’ boundaries of genes • lRNAs involved in regulating gene expression • Interleaved genome organization • Overlapping sense and antisense lRNA transcription • Protein coding genes • sRNAs at boundaries based on expression state • Unannotated nuclear transcription = precursors to sRNAs • Regulatory potential with non-protein coding sRNA and lRNA

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