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Two types of short RNAs flanking the 3’ ends of fly mRNAs

This study explores the presence of two types of short RNAs flanking the 3' ends of fly mRNAs through CAGE and Capped small RNAs analysis. CAGE data from 0-24h embryos and libraries from <200nt sRNAs in S2 cells were examined. The study reveals the distribution and characteristics of capped sRNAs and mono-P sRNAs at transcription start and termination sites.

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Two types of short RNAs flanking the 3’ ends of fly mRNAs

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  1. Two types of short RNAs flanking the 3’ ends of fly mRNAs ENCODE and modENCODE Consortia Meeting March 10-12, 2010 Delphine Fagegaltier Alexander Dobin Thomas Gingeras Cold Spring Harbor Laboratory

  2. Model in Human for generating capped long and small RNAs Long Capped RNAs Short Capped RNAs

  3. Survey of CAGE tags and capped sRNAs in flies • CAGE data from 0-24h embryos (Carninci Lab) (long capped RNAs from total RNAs) • Libraries from <200nt sRNAs in S2 cells (late embryonic tissue culture) • Capped sRNAs (CIP+TAP) • 5’mono-P sRNAs (no treatment) Selectively enriched for by enzymatic treatment: - Clonable -Non clonable 5’end of RNA after enzymatic treatment

  4. Fly sRNAs and CAGE at TSS and TTS First Exon Last Exon Transcription Start Site (TSS) Transcription Termination Site (TTS) CAGE (Capped long RNAs) Gene 5’Cap-sRNAs monoP-sRNAs

  5. Fly capped sRNAs at TTS come from PASRs of overlapping genes 5’CAP-sRNAs FILTERED ALL Gene TTS Gene TTS TSS Gene

  6. Fly mono-P sRNAs and CAGE at TTS ALL FILTERED CAGE monoP-sRNAs ALL FILTERED Gene TTS

  7. Summary • CAGE tags and capped small RNAs align across most part of the genes in fly, abundantly at the 5’ end of genes and in the internal exon regions. • However this relationship changes at the 3’ end of genes. • Near the TTS, we identify two classes of sRNAs: - Capped sRNAs downstream of the TTS, coming from overlapping 5’ends of genes (capped PASRs). - Uncapped 5’mono-P sRNAs . whose 3’ends align exactly with the TTS (sharp peak). . CAGE tags also accumulate at TTS (broader pattern). . 5’mono-P sRNAs come in part from overlapping 3’ and 5’ ends of neighboring genes; yet many persist at the TTS of genes with no overlapping TTS or TSS within 1kb. • At the TTS, we do not find a correlation in the stoechiometry of of CAGE and mono-P sRNAs reads.

  8. ModEncode Drosophila Transcriptome Group Peter Cherbas Lab Lucy Cherbas Dayu Zhang Piero Carninci Lab Timo Lassman Greg Hannon Lab Katalina Fejes-Toth Vihra Sotirova Assaf Gordon Michelle Rooks Tom Gingeras Lab Alexander Dobin (*Poster) Chenghai Xue Christopher Zaleski Philippe Batut Kimberly Bell Sudipto Chakrabortty Carrie Davis Jorg Drenkow Sonali Jha Inigo Lasa Wei Lin Johann Schlesinger Lei Hoon See Huaien Wang Thank you. + Poster tonight

  9. CAGE tags trimmed at 3' TTS TSS AAAAAA STAR will trim the 3' to map % of the trimmed CAGE tags % of the trimmed As Not a classic degradation pathway which would remove A-tails first

  10. Fly sRNAs and CAGE at TSS, IE, TTS First Exon Internal Exon (IE) Last Exon Transcription Start Site (TSS) Transcription Termination Site (TTS) CAGE Gene 5’Cap-sRNAs monoP-sRNAs

  11. 5’ mono-P-sRNAs are produced at the 3’ end of genes, whether or not (1.4K loci, C) a neighboring TSS (>2.5K loci, B) or TTS (>2.1K loci, B-C, S/AS pairs) from another gene is present <1kb away A B C 3.5K 1.4K Peak:6K SENSE no TSS or TTS <1kb away monoP-sRNAs All no TSS <1kb away Peak:0.7K 0.04K 0.5K ANTISENSE Gene Gene Gene TTS TTS

  12. CAGE tags are present at the 3’ end of genes with a TSS (6K S + 1K AS) or a TTS (5K S + 2K AS loci) <1kb away; (5K S + 1K AS) loci remain if no TSS or TTS lies within 1kb. Peak:10K Peak:5K Peak:16K SENSE CAGE no TSS or TTS <1kb away All no TSS <1kb away Peak:4K 3K 1K ANTISENSE Gene Gene Gene TTS TTS

  13. Fly sRNAs and CAGE at TTS SENSE CAGE m7G-sRNAs monoP-sRNAs ANTISENSE Gene Gene Gene TTS TTS TTS

  14. Fly sRNAs and CAGE at TTS(without reads <1kb away from another TSS) SENSE CAGE m7G-sRNAs mono-P-sRNAs ANTISENSE Gene Gene Gene TTS TTS TTS TSS

  15. Fly sRNAs and CAGE at TTS(without reads <1kb away from TTS OR TSS) SENSE CAGE m7G-sRNAs monoP-sRNAs ANTISENSE Gene Gene Gene TTS TTS TTS

  16. Length of capped sRNAs at TTS(mostly from neighboring TSSs (PASRs)). Window taken 200bp: -100 to +100b of TTS

  17. Length of mono-P sRNAs at TTS

  18. CAGE and mono-P sRNAs at TTS Read levels of CAGE and 5’ mono-P sRNAs per gene

  19. CAGE and mono-P sRNAs at TTS CAGE and mono-P sRNAs at TTS CAGE and capped sRNAs at TSS (more filtering required) Bulk= CAGE + capped sRNAs: Genes = ? (degraded, high Txn levels, sense antisense txn?…) capped sRNAs only Genes=? cannot be capped known sRNAs CAGE only, no or 1 capped sRNA Genes Initiating Txn only? CAGE may differ in 0-24h embryos versus S2 cells.

  20. CAGE and mono-P sRNAs at TTS CAGE and mono-P sRNAs at TTS per gene (per TTS, not gene, Window +-50bp Removed overlapping genes within 1kbp CAGE and mono-P sRNAs at TTS per gene (sum TTSs=1 gene) Window +-20bp Removed overlapping genes within 500bp Mixed (TAP) and mono-P sRNAs at TTS CAGE and capped sRNAs at TSS

  21. Distribution of capped RNAs (CAGE and 5’CAP-sRNAs)and 5’-mono-P sRNAs at TTS

  22. CAGE tags and capped small RNAs align at the TSS and IE in human cells. Affymetrix Nature000, 1-5 (2009) doi:10.1038/nature07759

  23. Enzymatic treatments(Clonable 5’ end RNA after enzymatic treatment) Products after treatment

  24. Method 1. RNAs <200nt 6. AAAAAA 2. - 2S rRNA depletion - 5’-end Enzymatic treatment to select for m7G-RNAs m7G-RNAs+ uncapped RNAs Mono P-RNAs 7. AAAAAA TTTTTT 8. QC, quantification qPCR 3. AAAAAAA 4. 9. Sequencing PE 76 AAAAAAA 5. AAAAAAA

  25. Selective enrichment for mono-P or capped sRNAs miRNAs snRNAs

  26. Back up info: • Number of genes with mono-P sRNAs ~ at TTS • Number of genes with CAGE ~ at TTS • Number of genes with capped sRNAs at TTS (all, can overlap <700)

  27. % of trimmed As at 3’ end in all combined TAP-treated C-tailed treated after removing overlapping genes, the same 2 plots you made for CAGE.

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