The alternative life of RNA: Sequencing meets single molecule approaches

RNA-Seq has provided valuable information about the transcriptomes from homogenized tissues or cultured cells. But as tissues consist of a heterogeneous pool of cells, specific cell-type differences in gene expression are diluted. Moreover, within the same population, individual cells show differences in gene expression. An adapted single-cell RNA-Seq (scRNA-Seq) method is providing new information about single cell transcriptomics. scRNA-Seq involves dissociation of the tissue and isolation of single cells, followed by a whole-transcriptome amplification that constructs sequencing libraries using cellular barcodes. This allows the processing of multiple cells in single reaction tubes. Latest improvements of the method were achieved by using microfluidic technologies in combination with molecular barcodes, increasing specificity while decreasing noise. Using this technique, not only different mRNA expression levels between distinct types of cells can be determined, but also the diversity of gene expression between developmental transitions. In order to characterize the length of 3’-UTRs in single cells, the application of scRNA-Seq has recently been improved. Researchers developed a barcoded, 3’-end specific sequencing (BAT-Seq) protocol where they combined unique cell barcodes with poly(dT) primers linked to unique molecular identifiers. BAT-Seq permitted to determine that alternative polyadenylation is highly variable across cell types, and that pluripotent populations express shorter 3’-UTRs. Recently, scRNA-Seq has been further improved to differentiate between individual dividing cells in mouse newborn brain. Others have developed Div-Seq, combining scRNA-Seq of nuclear transcripts (sNuc-Seq).

 Mechanisms to obtain alternative mRNA isoforms

rna-seq

(A) Transcript variability generated by alternative splicing. A.1) General alternative splicing (AS) of a gene containing three exons (E1, E2, E3) can yield two different mRNA isoforms by exclusion or inclusion of exon 2. A.2) During alternative splicing of last exons (ALE), the selection of either exon 2 (E2) or exon 3 (E3) as the last exon produces two distinct isoforms with different exonic sequences and 3’-untranslated regions (UTRs). A.3) Skipping of the splicing between exon 2 and 3 leads to intron retention (IR). The processed mRNA will contain an intronic sequence that is recognized by the exon junction complex (EJC) and may contain premature termination codons, resulting in nonsense-mediated decay (NMD). B) A 3’-UTR can harbor more than one polyadenylation site. The selection of proximal over distal polyadenylation sites, or reverse, will result in different 3’-UTR lengths, a process termed alternative polyadenylation (APA). Those sequences generated by intron retention or 3’-UTR lengthening can be recognized by trans-acting factors, e.g. RNA-binding proteins (RBP), or microRNAs (miRNAs), allowing for a more complex regulation of the alternative mRNA isoforms.

Fernández-Moya SM, Ehses J, Kiebler MA. (2017) The alternative life of RNA: Sequencing meets single molecule approaches. FEBS Lett [Epub ahead of print]. [article]

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