Small RNA sequences derived from pre-microRNAs in the supraspliceosome

Abstract

MicroRNAs (miRNAs) are short non-coding RNAs that negatively regulate the expression and translation of genes in healthy and diseased tissues. Herein, we characterize short RNAs from human HeLa cells found in the supraspliceosome, a nuclear dynamic machine in which pre-mRNA processing occurs. We sequenced small RNAs (<200 nt) extracted from the supraspliceosome, and identified sequences that are derived from 200 miRNAs genes. About three quarters of them are mature miRNAs, whereas the rest account for various defined regions of the pre-miRNA, and its hairpin-loop precursor. Out of these aligned sequences, 53 were undetected in cellular extract, and the abundance of additional 48 strongly differed from that in cellular extract. Notably, we describe seven abundant miRNA-derived sequences that overlap non-coding exons of their host gene. The rich collection of sequences identical to pre-miRNAs at the supraspliceosome suggests overlooked nuclear functions. Specifically, the abundant hsa-mir-99b may affect splicing of LINC01129 primary transcript through base-pairing with its exon-intron junction. Using suppression and overexpression experiments, we show that hsa-mir-7704 negatively regulates the level of the lncRNA HAGLR. We claim that in cases of extended base-pairing complementarity, such supraspliceosomal pre-miRNA sequences might have a role in transcription attenuation, maturation and processing.

MiR&moRe2: A Bioinformatics Tool to Characterize microRNAs and microRNA-Offset RNAs from Small RNA-Seq Data

“…In accordance with previous reports [11,20,29], moRNAs were more abundant in the nuclear fraction of cellular RNA. Further, moRNAs were generally less expressed than miRNAs, but specific moRNAs were abundant, even more than the flanking miRNA [11], representing an alternative product respective to the mature miRNA from the same hairpin arm.…”

“…In the first study (ASI) [14] moRNAs were specifically reported along with their expression estimates. In contrast, the second work (BUR) [20] detected moRNAs and reported them using conventional naming [43]; whereas in MAV [29] moRNAs were referred with a custom denomination. Straightforward comparisons were not possible since each work applied its own custom discovery and analysis pipeline, which, in addition, were based on currently outdated miRNA annotation.…”

“…Straightforward comparisons were not possible since each work applied its own custom discovery and analysis pipeline, which, in addition, were based on currently outdated miRNA annotation. Moreover, the authors did not provide automated software to replicate their analysis, and, in one case [29], they used custom naming to refer to moRNAs. These issues would not have arisen if the authors could have used an automated bioinformatics pipeline such miR&moRe2.…”

“…To evaluate miR&moRe2 performance, we applied our method to three datasets from previous studies on human cell samples in which (i) moRNAs were detected, validated and studied (ASI dataset) [14], (ii) moRNAs were considered and explicitly mentioned (BUR dataset) [20], and (iii) sequences derived from miRNA precursor flanking regions were detected (MAV dataset) [29] (Table 1). Considering the datasets together,~8% of miRNA-expressing precursors expressed at least one moRNA.…”

“…Friedländer and colleagues [30] validated their prediction method by analyzing the expression pattern of the predicted pre-miRNAs and miRNAs upon silencing of the miRNA biogenesis pathway [14]; (b) correlation of moRNA expression estimates (read counts) between miR&moRe2 predictions and values in [14] for each sample of the dataset (human embryonic stem cell lines HS181, HS401 and H9, plus fibroblast HFF-1). H9 d00/d15: day 0 and day 15 of H9 differentiation as detailed in [14]; (c) moRNAs reported in [20] compared to miR&moRe2 moRNAs predicted from the BUR dataset; (d) miR&moRe2 moRs and new miRs predicted from the MAV dataset, compared to miRNA gene aligned sequences grouped as in [29] (overlap-regions, undefined complement and extension sequences).…”