Whole-genome expression analysis of mammalian-wide interspersed repeat elements in human cell lines

Abstract

With more than 500,000 copies, mammalian-wide interspersed repeats (MIRs), a sub-group of SINEs, represent ∼2.5% of the human genome and one of the most numerous family of potential targets for the RNA polymerase (Pol) III transcription machinery. Since MIR elements ceased to amplify ∼130 myr ago, previous studies primarily focused on their genomic impact, while the issue of their expression has not been extensively addressed. We applied a dedicated bioinformatic pipeline to ENCODE RNA-Seq datasets of seven human cell lines and, for the first time, we were able to define the Pol III-driven MIR transcriptome at single-locus resolution. While the majority of Pol III-transcribed MIR elements are cell-specific, we discovered a small set of ubiquitously transcribed MIRs mapping within Pol II-transcribed genes in antisense orientation that could influence the expression of the overlapping gene. We also identified novel Pol III-transcribed ncRNAs, deriving from transcription of annotated MIR fragments flanked by unique MIR-unrelated sequences, and confirmed the role of Pol III-specific internal promoter elements in MIR transcription. Besides demonstrating widespread transcription at these retrotranspositionally inactive elements in human cells, the ability to profile MIR expression at single-locus resolution will facilitate their study in different cell types and states including pathological alterations.

MIR sequences recruit zinc finger protein ZNF768 to expressed genes

“…MIRs are ∼240 bp long and consist of tRNA-derived sequences, a 70 bp MIR-specific core region, and sequences similar to the 3′ ends of LINEs. MIRs are enriched at gene loci in euchromatin, harbor putative transcription-factor binding sites, provide insulator and enhancer function (5–8), encode microRNAs, are transcribed by RNA polymerase III (9,10), are associated with tissue-specific gene expression (5,11), and sometimes provide splicing signals and contribute to exonization (12). MIRs constitute 5–16% of the genome in marsupials and monotremes and 0.5-3% in placentalia (13).…”

Identification of RNA Polymerase III-Transcribed SINEs at Single-Locus Resolution from RNA Sequencing Data

“…In particular, both left and right SINE fragments could be a source of Pol III-derived chimeric transcripts originating from an upstream or ending in a downstream unique SINE-unrelated moiety. Indeed, while left SINE fragments contain Pol III promoters, right ones could still potentially be a target of the Pol III machinery complex if their upstream SINE-unrelated regions provide A- and B-box sequences that, for example, have been preserved from the mutation of an ancestral (now degenerated) SINE portion [ 18 , 19 ].…”

“…Our bioinformatics pipeline [ 19 ] is outlined in Figure 2 , and consists of three main steps: (1) genome expression coverage vector creation; (2) global alignment between the annotated element and its corresponding full-length sequence; (3) application of a filter aimed at excluding passenger SINE transcripts.…”

“…The unique genomic region downstream of the 3’ end of the MIR element (dashed line) could contain the Pol III termination signal (T n ) which may be located at varying distances from the end of the MIR body. Adapted from [ 18 , 19 ].…”

“… Bioinformatic pipeline for SINE RNA expression analysis at single locus resolution. Shown is a flow-diagram of a recently developed bioinformatic pipeline for the identification of autonomously expressed SINE loci from RNA-seq data sets [ 18 , 19 ]. See text for details.…”