Two distinct mechanisms generate endogenous siRNAs from bidirectional transcription in Drosophila melanogaster - PubMed (original) (raw)

Two distinct mechanisms generate endogenous siRNAs from bidirectional transcription in Drosophila melanogaster

Katsutomo Okamura et al. Nat Struct Mol Biol. 2008 Jun.

Erratum in

Abstract

Cis-natural antisense transcripts (cis-NATs) have been speculated to be substrates for endogenous RNA interference (RNAi), but little experimental evidence for such a pathway in animals has been reported. Analysis of massive Drosophila melanogaster small RNA data sets now reveals two mechanisms that yield endogenous small interfering RNAs (siRNAs) via bidirectional transcription. First, >100 cis-NATs with overlapping 3' exons generate 21-nt, and, based on previously published small RNA data [corrected] Dicer-2 (Dcr-2)-dependent, 3'-end modified siRNAs. The processing of cis-NATs by RNA interference (RNAi) seems to be actively restricted, and the selected loci are enriched for nucleic acid-based functions and include Argonaute-2 (AGO2) itself. Second, we report that extended intervals of the thickveins and klarsicht genes generate exceptionally abundant siRNAs from both strands. These siRNA clusters derive from atypical cis-NAT arrangements involving introns and 5' or internal exons, but their biogenesis is similarly Dcr-2- and AGO2-dependent. These newly recognized siRNA pathways broaden the scope of regulatory networks mediated by small RNAs.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Distinct size distributions of different small RNA–generating loci. Each graph plots the number of small RNA reads on the y axis and the length of small RNAs on the x axis. (a) 93 ribosomal protein (RpL) genes. (b) 131 canonical miRNA genes; miRNA reads are blue and miRNA* reads are red. (c) The two most highly expressed hpRNA genes, hp-CG18854 and hp-CG4068 (ref. 14). (d) white siRNAs cloned from GMR-whiteIR heads; sense reads are blue and antisense reads are red. (e) GFP siRNAs cloned from S2 cells carrying a GFP transgene and transfected with GFP dsRNA; sense reads are blue and antisense reads are red. (f) All 5′ _cis_-NAT loci except the _tkv/CG14033 cis_-NAT. The combined overlap reads from both strands are blue, and the combined non-overlap exonic reads from both strands are red. (g) The non-overlap regions of all non-siRNA, 3′ _cis_-NAT loci, except the _hp-CG18854/IP3K cis_-NAT. In this and subsequent graphs, top-strand reads are blue and bottom-strand reads are red. (h) The overlap regions of 117 3′ _cis_-NAT siRNA loci. (i) The tkv-RC/CG14033 siRNA cluster. (j) The klar siRNA clusters.

Figure 2

Figure 2

The overlap regions of a subset of 3′ _cis_-NAT loci preferentially generate siRNAs. (a) Small RNAs mapped to the _AGO2/CG7739 cis_-NAT via the UCSC genome browser. Small RNAs from both strands mapped nearly exclusively to the mRNA-overlap region, and the vast majority (> 75%) of these are 21 nt in length (Supplementary Table 1). (b) A subset of 3′ _cis_-NAT overlaps are associated with particularly elevated siRNA density.

Figure 3

Figure 3

Most coexpressed 3′ _cis_-NAT pairs do not generate siRNAs. Gene expression in duplicate isolates of S2 cell RNA was assessed using Affymetrix arrays. For each _cis_-NAT pair, we recorded the lower gene expression value; thus, the transcripts in a given pair have expression that is equal to or higher than the value plotted on the _x_-axis. The vast majority of _cis_-NAT siRNA loci include pairs in which both genes are called ‘present’ in S2 cells. However, many more non-siRNA _cis_-NATs involve gene pairs for which both members are called ‘present’ in S2 cells.

Figure 4

Figure 4

The thickveins (tkv) and klarsicht (klar) loci define a distinct class of exceptionally abundant siRNAs derived from bidirectional transcription. (a) The tkv locus covers > 50 kb and comprises several alternatively spliced transcripts. The CG14033 transcript is generated from the strand opposite to tkv, and its promoter resides between _tkv_-RB and tkv-RC. A dense cluster of siRNAs maps to the second half of CG14033 and overlaps the short tkv-RC coding exon on the other strand. (b) The klar locus extends over 100 kb and comprises several alternatively spliced transcripts. Two massive clusters of siRNAs derive from both strands of an intron; the 4.4-kb cluster overlaps two short klar coding exons. Note klar primary transcription across the CG32467/CG32468 inverted gene pair, which theoretically adopts an extended hairpin structure (Supplementary Fig. 4), does not generate siRNAs. At the bottom, a magnified view of klar exons that overlap the 4.4-kb cluster shows that siRNA production in this region is clearly related to klar exons.

Figure 5

Figure 5

Biogenesis and structural features of klar siRNAs. (a) We carried out northern analysis on RNA isolated from S2 cells treated with the indicated dsRNAs. Quantification with the hybridized 2S signal across three northern experiments showed 27% klar siRNAs in AGO2 knockdown cells, 20% in Dcr-2 knockdown cells and 58% in loqs knockdown cells. (b) 5′ end and 3′ end analysis. S2 cell RNA was β-eliminated to probe 3′ ends and treated with phosphatase to probe 5′ ends. Compared to untreated RNA (Ø), most miR-8 shifts to a faster-migrating species following β-elimination (β), but klar siRNAs are resistant, indicating 3′ blockage. The slight reduction in mobility of miR-8 and klar siRNAs following phosphatase (CIP) treatment indicates 5′ phosphates.

Comment in

Similar articles

Cited by

References

    1. Zhang Y, Liu XS, Liu QR, Wei L. Genome-wide in silico identification and analysis of cis natural antisense transcripts (cis-NATs) in ten species. Nucleic Acids Res. 2006;34:3465–3475. - PMC - PubMed
    1. Katayama S, et al. Antisense transcription in the mammalian transcriptome. Science. 2005;309:1564–1566. - PubMed
    1. Yelin R, et al. Widespread occurrence of antisense transcription in the human genome. Nat. Biotechnol. 2003;21:379–386. - PubMed
    1. Lipman DJ. Making (anti)sense of non-coding sequence conservation. Nucleic Acids Res. 1997;25:3580–3583. - PMC - PubMed
    1. Borsani O, Zhu J, Verslues PE, Sunkar R, Zhu JK. Endogenous siRNAs derived from a pair of natural cis-antisense transcripts regulate salt tolerance in Arabidopsis. Cell. 2005;123:1279–1291. - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources