melRNA-seq for Expression Analysis of SINE RNAs and Other Medium-Length Non-Coding RNAs

Abstract

Background Small interspersed elements (SINEs) are transcribed by RNA polymerase III (Pol III) to produce RNAs typically 100–500 nucleotides in length. Although their RNA abundance can be evaluated by Northern blotting and primer extension, the nature (sequence, exact length, and genomic origin) of these RNAs cannot be revealed by these methods. Moreover, mRNA sequencing (mRNA-seq) is not able to distinguish bona fide SINE RNAs or SINE sequences present in longer transcripts. Results To elucidate the abundance, source loci, and sequence nature of SINE RNAs, we established a deep sequencing method, designated as melRNA-seq (medium-length RNA-seq), which can determine whole-length RNA sequences. Total RNA samples were treated with 5′ pyrophosphohydrolase (RppH), which allowed ligation of an RNA adaptor to the 5′ end of intact SINE RNAs. Similarly, another adaptor was ligated to the 3′ end, followed by reverse transcription, PCR amplification, size selection, and single-end deep sequencing. The analysis of two biological replicates of RNAs from mouse spermatogonia showed high reproducibility of SINE expression data both at family and locus levels. Conclusions This new method can be used for quantification and detailed sequence analysis of medium-length non-coding RNAs, such as rRNA, snRNA, tRNAs, and SINE RNAs. Further, its dynamic range is much wider than Northern blotting and primer extension.

The Pol III transcriptome: Basic features, recurrent patterns, and emerging roles in cancer

“…Like tRNA, nascently transcribed precursor 5S rRNA is bound by the chaperone protein La through recognition of its 3’-oligo(U) tract. Though our current understanding of human 5S rRNA processing and maturation remains limited, relevant studies in Drosophila suggest 5S rRNA undergoes 3’-exonucleolytic maturation via REXO5, a conserved 3’–’ RNA exonuclease ( Gerstberger et al, 2017 ), generating a mature 119-nucleotide rRNA that largely retains its 5’-PPP moiety ( Mori & Ichiyanagi, 2021 ). In contrast to tRNA, current evidence suggests 5S rRNA is largely devoid of post-transcriptional modifications ( Natchiar et al, 2017 ; Taoka et al, 2018 ).…”

The sixth Japanese meeting on biological function and evolution through interactions between hosts and transposable elements

“…In contrast to the expression analysis of long interspersed elements (LINEs) and LTR retrotransposons, the expression of short interspersed elements (SINEs), which are transcribed by RNA Pol III and are typically < 500-bp long, is difficult to analyze by deep sequencing because many copies reside within other transcripts such as mRNAs. Kenji Ichiyanagi (Nagoya University, Japan) recently established a deep sequencing method, melRNA-seq, to determine the RNA abundance, RNA length, and source loci of SINEs [ 32 , 33 ]. They analyzed the expression of Alu SINE in human cells which revealed a 9-fold increase in Alu expression after heat shock treatment.…”