CapSeq and CIP-TAP Identify Pol II Start Sites and Reveal Capped Small RNAs as C. elegans piRNA Precursors

Abstract

SUMMARY Piwi-interacting (pi) RNAs are germline-expressed small RNAs linked to epigenetic programming. C. elegans piRNAs are thought to be transcribed as independent gene-like loci. To test this idea and to identify potential Transcription Start (TS) sites for piRNA precursors, we developed CapSeq, an efficient enzymatic method for 5′-anchored RNA profiling. Using CapSeq we identify candidate TS sites, defined by 70–90 nt sequence tags, for over 50% of annotated Pol II loci. Surprisingly, however, these CapSeq tags failed to identify the overwhelming majority of piRNA loci. Instead, we show that the likely piRNA precursors are ~26 nt capped-small (cs) RNAs that initiate precisely 2 nt upstream of mature piRNAs, and that piRNA processing or stability requires a U at the csRNA +3 position. Finally, we identify a heretofore-unrecognized class of piRNAs processed from csRNAs that are expressed at promoters genome wide, nearly doubling the number of piRNAs available for genome surveillance.

RNA Polymerase II transcription elongation and Pol II CTD Ser2 phosphorylation

“…147,148 Thus, although the TSS has been mapped for individual genes in C. elegans, 149 genomewide approaches for TSS identification have only recently been performed. 145,147,148,150 These recent results have, for the first time, allowed scientists to analyze the regulation of early steps of Pol II transcription on a global scale. CTD phosphorylation patterns in C. elegans are similar to other metazoans.…”

“…C. elegans has been a unique model for understanding transcription regulation because it is one of the few well-studied models that has transsplicing of Pol II transcripts. Trans-splicing, which occurs on ~70% of C. elegans genes, 144,145 involves the replacement of the 5′ end of transcripts with a short, 21-22 nucleotide RNA sequence, called a splice leader. 146 Because trans-splicing occurs on the majority of genes, the TSS for many Pol II genes cannot be mapped from the 5′ sequence of mature mRNA as is commonly done in other organisms.…”

End Sequence Analysis Toolkit (ESAT) expands the extractable information from single-cell RNA-seq data

“…DGE libraries have obvious advantages over full-length RNA-seq libraries: They work well for low-quality RNA; PCR duplicates arising during amplification are easily detected by using molecular indices; and since each mRNA molecule is represented by a single tag, quantification is greatly simplified (Asmann et al 2009;Matsumura et al 2010;Shiroguchi et al 2012;Kawaji et al 2014). While the simple library construction by poly(A) selection or priming has made sequencing the 3 ′ end of transcripts the most common approach for DGE, 5 ′ sequencing is also a viable strategy for DGE, and several methods exist that take advantage of the 5 ′ cap that protects eukaryotic mRNAs to build libraries that target the start of transcripts rather than their ends (Gu et al 2012;Takahashi et al 2012).…”

Overview of gene structure in C. elegans

“…Even the full extent of the primary transcript is frequently not known because a majority (70%) of protein-coding genes are rapidly modified by trans-splicing, which involves the addition of a short 22 nt exogenous RNA species to the 5' end of a transcript (Zorio et al, 1994). Definition of the true 5' ends of genes is an active area of research (Chen et al 2013;Kruesi et al 2013;Saito et al 2013;Gu et al 2012). Some non-coding RNA genes are also trans-spliced; a precursor of the microRNA let-7 (C05G5.6) was identified with a trans-splice leader sequence (Bracht et al, 2004).…”