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Ähnlich wie linc-HOXA1 is a non-coding RNA that represses Hoxa1 in cis
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linc-HOXA1 is a non-coding RNA that represses Hoxa1 in cis
- 1. linc-HOXA1 is a non-coding RNA that represses Hoxa1 transcription in cis Hédia Maamar1, Moran Cabili2, John Rinn2, Arjun Raj1 1University of Pennsylvania 2Harvard University and Broad Institute Genes and Development, in press (2013) Hédia Maamar
- 2. Xist is a non-coding RNA that regulates transcription across an entire chromosome Xist RNA
- 3. Hybridization of multiple oligonucleotides yields fluorescent spots corresponding to single RNAs Target mRNATarget mRNA Multiple singly-labeled DNA oligonucleotides, 20 bases each Raj et al. Nature Methods, 2008 RNA fluorescence in situ hybridization (RNA FISH) Femino et al. Science 1998
- 4. The Hox clusters harbor several non-coding RNAs phenomena, polycistronism and antisense transcription. Polycistronic clusters A polycistronic cluster designates two or more genes co- transcribed from a single promoter, sharing a non-coding exon, and whose products are generated by alternative splicing [18]. An operon is a particular case where the mRNA retains the different products after splicing. In Mammals, both operons and polycistronic clusters are scarcely documented [18]. One clear example nonetheless, is the case of the Hoxc4, Hoxc5 and Hoxc6 Widespread antisense transcription Our analysis also revealed the existence of 15 TUs distinct from the Hox and mir genes that are poly-adenylated and alternatively spliced like genuine products of RNA Polymerase II. Most of these TUs (14/15) are transcribed antisense (AS) to Hox genes (see fig 2), and AS transcription can represent up to 38% of the spliced transcripts (38.46% for HoxA, 33.11% for HoxB, 13.16% for HoxC and 34.84% for HoxD). Cis-encoded antisenses and bidirectional promoters are now known to be abundant in the Figure 2. Synopsis of transcriptional activity in the Human Hox Clusters. Sense and antisense transcriptions are in red and green respectively. Dark and light shaded boxes represent exon and intron. Mir genes are in blue. The three long transcripts presenting a murine homolog with a conserved exon-exon boundary are headed by **. The 13 TUs (12 antisense and one sense) located at similar position in human and mouse are denoted by an asterisk (*) while the 10 TUs showing conservation are depicted with doi:10.1371/journal.pone.0000356.g002 Rinn et al. Cell 2007 Mainguy et al. PLOS ONE 2007
- 5. linc-HOXA1 is a lincRNA expressed in mouse ES cells located near Hoxa1 exon 3c exon 3b exon 3a exon 1 exon 2 Chr6 11.5 kb90 kb 41 kb 3 kb Isoform 1 Isoform 2 Isoform 3 Skap2 Hoxa1 Hoxa2 Hoxa3linc-HOXA1
- 6. There are three isoforms of linc-HOXA1 in mouse embryonic stem cells exon 3c exon 3b exon 3a exon 1 exon 2 Isoform 1 Isoform 2 Isoform 3 Isoform 1 Isoform 2 Isoform 3 exon 3c exon 3b exon 3a linc-HOXA1
- 7. Expression of the three isoforms of linc-HOXA1 was highly correlated in single cells exon 3c exon 3b exon 3a exon 1 exon 2 Isoform 1 Isoform 2 Isoform 3 Isoform 1 Isoform 2 Isoform 3 exon 3c exon 3b exon 3a linc-HOXA1 120 120 90 90 60 60 E3a molecules per cell E3bmoleculespercell E3cmoleculespercell E3cmoleculespercell30 30 0 0 1209060 E3a molecules per cell 300 1209060 E3b molecules per cell 300 y = ax + b a = 1.296 [1.084, 1.507] b = 4.893 [2.568, 7.219] y = ax + b a = 2.031 [1.750, 2.312] b = 9.202 [6.112, 12.29] y = ax + b a = 1.253 [1.095, 1.411] b = 6.652 [3.552, 9.753]
- 8. linc-HOXA1 and Hoxa1 transcripts both express in mouse embryonic stem cells MeannumberofRNApercell A B linc-HOXA1 linc-HOXA1 transcription siteHoxa1
- 9. linc-HOXA1 appears to correlate with Hoxa1 at the population level 0 0 654321 20 linc-HOXA1 Hoxa1 40 Number of days of retinoic acid expos 0 days retinoic acid exposure 1 day retinoic acid exposure 2 da acid MeannumberofRNApercell 180 180 A C B linc-HOXA1 linc-HOXA1 transcription siteHoxa1 Threshold 0 0 9654321 20 linc-HOXA1 Hoxa1 40 Number of days of retinoic acid exposure 0 days retinoic acid exposure 1 day retinoic acid exposure 2 days retinoic acid exposure MeannumberofRNApercell 40 60 120 180 120 180 Hoxa1mRNA A C B linc-HOXA1 linc-HOXA1 transcription siteHoxa1 Hoxa1 linc-HOXA1 125 26 127 35 n = 313, p = 0.33 Hoxa1 linc-HOXA1 26 107 1 101 n = 235, p = 10-5 Hoxa1 linc-HOXA1 254 56 43 1 n = 354, p = 0.007 Threshold
- 10. But linc-HOXA1 and Hoxa1 RNA anti-correlate at the single cell level 0 0 40 80 120 linc-HOXA1 RNA 0 days retinoic acid exposure 1 day retino acid exposu 0 40 linc-HOXA1 R Mean 0 20 40 60 0 60 120 180 Hoxa1mRNA C n Hoxa1 linc-HOXA1 26 107 1 101 n = 235, p = 10-5 Threshold Threshold
- 11. Treatment with retinoic acid eliminates this anti- correlation 0 0 9654321 20 Hoxa1 Number of days of retinoic acid exposure 0 40 80 120 linc-HOXA1 RNA 0 days retinoic acid exposure 1 day retinoic acid exposure 2 days retinoic acid exposure 0 40 80 120 linc-HOXA1 RNA 0 40 80 120 linc-HOXA1 RNA MeannumberofRNAp0 20 40 60 0 60 120 180 0 60 120 180 Hoxa1mRNA C Hoxa1 linc-HOXA1 125 26 127 35 n = 313, p = 0.33 Hoxa1 linc-HOXA1 26 107 1 101 n = 235, p = 10-5 Hoxa1 linc-HOXA1 254 56 43 1 n = 354, p = 0.0077 Threshold Threshold
- 12. We successfully knocked down linc-HOXA1 with both Isis anti-sense oligonucleotides and siRNA NumberofRNAmoleculespercell controlknockdow n controlknockdow n controlknockdow n controlknockdow n 12 16 Isis knockdown siRNA knockdown linc-HOXA1 knockdown linc-HOXA1 Hoxa1 8 4 0 linc-HOXA1 Hoxa1
- 13. But only the antisense oligonucleotides showed an effect on Hoxa1... why? NumberofRNAmoleculespercell controlknockdow n controlknockdow n controlknockdow n controlknockdow n 12 16 Isis knockdown siRNA knockdown linc-HOXA1 knockdown linc-HOXA1 Hoxa1 8 4 0 linc-HOXA1 Hoxa1
- 14. We looked to changes to transcription sites after linc-HOXA1 knockdown linc-HOXA1 transcription site
- 15. Direct transcript visualization reveals that linc- HOXA1 regulates Hoxa1 at the site of transcriptionSupplementary Figure 5 DAPI Control (scramble knockdown) linc-HOXA1 knockdown siRNAIsisanti-senseoligonucleotides DAPIlinc-HOXA1 exon linc-HOXA1 exon
- 16. Direct transcript visualization reveals that linc- HOXA1 regulates Hoxa1 at the site of transcriptionSupplementary Figure 5 DAPI Control (scramble knockdown) linc-HOXA1 knockdown siRNAIsisanti-senseoligonucleotides DAPIlinc-HOXA1 exon linc-HOXA1 exon
- 17. Transcription site knockdown accounts for the discrepancy between knockdown methods NumberofRNAmoleculespercell controlknockdow n controlknockdow n controlknockdow n controlknockdow n 12 16 Isis knockdown siRNA knockdown linc-HOXA1 knockdown linc-HOXA1 Hoxa1 8 4 0 linc-HOXA1 Hoxa1
- 18. Transcription site knockdown accounts for the discrepancy between knockdown methods NumberofRNAmoleculespercell controlknockdow n controlknockdow n controlknockdow n controlknockdow n 12 16 Isis knockdown siRNA knockdown linc-HOXA1 knockdown linc-HOXA1 Hoxa1 8 4 0 linc-HOXA1 Hoxa1 Number of active transcription sites per cell Number of active transcription sites per cell 0.4 0.2 0 0.2 0
- 19. Over-expression of linc-HOXA1 did not affect Hoxa1 expression levels 40 linc-HOXA1 isoforms Hoxa1 mRNA Numberoflinc-HOXA1 RNAmolecules 60 20 0 NumberofHoxa1 mRNAmolecules 20 10 0 G FP (control) Isoform 1 Isoform 2Isoform 3 G FP (control) Isoform 1 Isoform 2Isoform 3
- 20. Over-expression of linc-HOXA1 did not affect Hoxa1 expression levels Hoxa1mRNAmolecules Isoform 1 0 150 300 Hoxa1 linc-HOXA1 17 64 15 58 n = 154, p = 0.95 Isoform 2 0 150 300 Hoxa1 linc-HOXA1 8 40 19 77 n = 144, p = 0.65 linc-HOXA1 RNA molecules linc-HOXA1 RNA molecules linc-HOXA1 RNA molecules linc-HOXA1 RNA molecules Isoform 3 0 150 300 Hoxa1 linc-HOXA1 7 24 21 92 n = 144, p = 0.62 40 80 0 GFP (control) 0 30 60 Hoxa1 linc-HOXA1 26 112 6 9 n = 153, p = 0.055 Threshold
- 21. We pulled down proteins associated with linc- HOXA1 and analyzed by mass-spectrometry Biotinylated RNA PAGE Mass spectrometry Cell lysate
- 22. We pulled down proteins associated with linc- HOXA1 and analyzed by mass-spectrometry Biotinylated RNA PAGE Mass spectrometry Cell lysate exon3c anti-sense exon3c sense exon3a,b anti-sense exon3a,b sense exon3a sense 30 40 50 80 PURB (mass-spectrometry) exon 3c exon 3b exon 3a exon 1 exon 2 linc-HOXA1 GAA-rich region
- 23. We pulled down PURB (with a Myc tag) and looked for linc-HOXA1 RNA by RT-PCR anti-Myc PURB-Myc RT-PCR 500 bp W ater(negative control) TotallysateIgG R N A pulldow n M YC R N A pulldow n 200 bp
- 24. Knocking down PURB increased Hoxa1 levels and got rid of the anti-correlationNumberofRNAmoleculespercell Purb siRNA knockdown linc-HOXA1 Hoxa1Purb 15 25 20 10 5 0 controlknockdow n controlknockdow ncontrolknockdow n Purb siRNA knockdown 0 60 Threshold 120 linc-HOXA1 RNA 0 20 40 60 Hoxa1mRNA Hoxa1 linc-HOXA1 12 3 23 6 n = 44, p = 0.96
- 25. What lessons does linc-HOXA1 hold for lincRNA function? linc-HOXA1 transcription site