Pek JW et al. (2015), Stable intronic sequence RNAs have possible reg...

XB-ART-51463

J Cell Biol 2015 Oct 26;2112:243-51. doi: 10.1083/jcb.201507065.

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Stable intronic sequence RNAs have possible regulatory roles in Drosophila melanogaster.

Pek JW , Osman I , Tay ML , Zheng RT .

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Stable intronic sequence RNAs (sisRNAs) have been found in Xenopus tropicalis, human cell lines, and Epstein-Barr virus; however, the biological significance of sisRNAs remains poorly understood. We identify sisRNAs in Drosophila melanogaster by deep sequencing, reverse transcription polymerase chain reaction, and Northern blotting. We characterize a sisRNA (sisR-1) from the regena (rga) locus and show that it can be processed from the precursor messenger RNA (pre-mRNA). We also document a cis-natural antisense transcript (ASTR) from the rga locus, which is highly expressed in early embryos. During embryogenesis, ASTR promotes robust rga pre-mRNA expression. Interestingly, sisR-1 represses ASTR, with consequential effects on rga pre-mRNA expression. Our results suggest a model in which sisR-1 modulates its host gene expression by repressing ASTR during embryogenesis. We propose that sisR-1 belongs to a class of sisRNAs with probable regulatory activities in Drosophila.

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Species referenced: Xenopus tropicalis
Genes referenced: myc rps27

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	Figure 1. Identification of sisRNAs. (A) Diagram of an ovariole showing stages in the development of the oocyte and its accompanying nurse cells. Also shown is a 0â2-h embryo. GV, germinal vesicle. (B) RT-PCR showing expression of some sisRNAs in unfertilized eggs. (C) RT-PCR showing expression of some RNase R-resistant sisRNAs in unfertilized eggs. rga exon was used as a positive control for RNase R activity. (D) Northern blots showing expression of mbt, csp, and RpS27 sisRNAs during development and in adult somatic tissues and gonads. The gels were stained with SYBR Gold to visualize 5S rRNA as a loading control. L1, first instar larvae; L2, second instar larvae; L3, third instar larvae.
	Figure 2. sisRNA (sisR-1) from the regena locus. (A) Genome browser view of exonic and intronic sequences in the rga locus. Blue and red arrows indicate exonic and intronic primers used for RT-PCR. The bottom panel shows an enlarged view of the intronic peak. Red bars indicate the regions to which probes A and B hybridize. (B) Strand-specific RT-PCR using primers in the intron and exon of rga. (C) Northern blots using probes A and B to detect expression of sisR-1 in 0â2 h embryos and ovaries are shown. A band of â¼300 nt was detected with probe A (red arrows). Probe B gave no detectable signal in RNA from 0â2-h embryos but did hybridize with 10 pg of in vitro transcribed (IVT) RNA. (D) Northern blots showing expression of sisR-1 during development and in adult somatic tissues and gonads. The gels were stained with SYBR Gold to visualize 5S rRNA as a loading control. L1, first instar larvae; L2, second instar larvae; L3, third instar larvae.
	Figure 3. Processing of sisR-1. (A) Diagram showing the insertion of an EP element EY10731 used to overexpress the rga pre-mRNA. L, long; S, short. (B) A Northern blot showing expression of sisR-1 in the ovaries of controls versus rga overexpression flies. RT-PCR showing expression of rga mRNA and actin5C in the ovaries of controls versus rga overexpression flies. (C) Diagram showing the construct used to overexpress rga intronic sequences. (D) Northern blot showing expression of sisR-1 in ovaries of controls versus rga intronic sequence overexpression flies. RT-PCR verifies expression of dsRed mRNA in the ovaries of dsRed-intron-myc overexpression flies but not in controls. (E) Northern blots showing expression of sisR-1 in the controls versus ldbr RNAi third instar larvae. (F) Model for processing of sisR-1 from the rga pre-mRNA. (G) Northern blot showing detection of sisR-1 in both the nuclear (N) and cytoplasmic (C) fractions of 14â24-h embryos. RT-PCR showing the expression of U85 only in the nuclear fraction. EtBr staining showing the enrichment of 18S, 28S rRNA in the cytoplasmic fraction. RNAs were run on the same gel, and the intervening lanes were removed (white vertical lines). (H) Diagram showing the 5â² and 3â² ends of the nuclear and cytoplasmic sisR-1 determined by RACE. Question mark depicts unknown 3â² end of cytoplasmic sisR-1. (I) Predicted structure of nuclear sisR-1 by Vienna RNA fold software. Red dotted region is shown in J. (J) Magnified view of the dotted region in I shows the base pairing. (K) Diagram with red arrows to indicate regions where the primers anneal to. (L) RT-PCR showing the detection of the short, but not long, isoform of sisR-1 in the cytoplasmic fraction. (M) Model showing the processing of nuclear sisR-1 to cytoplasmic sisR-1 by 3â² end processing.
	Figure 4. ASTRpromotes robust expression of rga pre-mRNA. (A) Genomic locus showing regions of rga, sisR-1, and ASTR. Red bar indicates the region where the ASTR probe binds. Red arrowhead points to a putative branch point (BP) CTAAT. (B) Northern blots showing expression of ASTR in 2â14-h embryos. In vitro transcribed (IVT) RNA was used as positive controls. The gels were stained with EtBr to visualize 18S, 28S rRNA as a loading control. (C) Strand-specific RT-PCR detecting the presence of ASTR in the nuclear but not the cytoplasmic fraction of 2â14-h embryos. RT-PCR showing the high expression of U85 in the nuclear fraction. EtBr staining showing the enrichment of 18S, 28S rRNA in the cytoplasmic fraction. (D) Northern blot showing the expression of ASTR in controls versus da-Gal4>ASTR RNAi 2â14-h embryos. The gel was stained with EtBr to visualize 18S, 28S rRNA as a loading control. (E) qPCR data showing relative expression of rga pre-mRNA normalized to actin5C in the controls versus da-Gal4>ASTR RNAi 2â14-h embryos. Error bars represent SD. n = 3. P-value represents t test.
	Figure 5. sisR-1promotes robust repression of ASTR. (A) Northern blot showing the expression of ASTR in controls versus da-Gal4>sisR-1 2â14-h embryos. (B) qPCR data showing relative expression of rga pre-mRNA normalized to actin5C in the controls versus da-Gal4>sisR-1 2â14-h embryos. Error bars represent SD. n = 3. P-value represents t test. (C) Northern blots showing expression of ASTR in controls versus embryos expressing sisR-1 shRNA. The gels were stained with EtBr to visualize 18S, 28S rRNA as a loading control. (D) qPCR data showing fold change of rga pre-mRNA expression (normalized to actin5C) from 2â14-h to 14â24-h embryos in controls versus sisR-1 RNAi versus sisR-1 RNAi + ASTR RNAi embryos. Error bars represent SD. n = 3. P-value represents t test. (E) Model for the regulatory relationship between sisR-1, ASTR, and rga pre-mRNA during embryogenesis.

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