Single-cell RNA cap and tail sequencing (scRCAT-seq) reveals subtype-specific isoforms differing in transcript demarcation

Abstract

The differences in transcription start sites (TSS) and transcription end sites (TES) among gene isoforms can affect the stability, localization, and translation efficiency of mRNA. Gene isoforms allow a single gene diverse functions across different cell types, and isoform dynamics allow different functions over time. However, methods to efficiently identify and quantify RNA isoforms genome-wide in single cells are still lacking. Here, we introduce single cell RNA Cap And Tail sequencing (scRCAT-seq), a method to demarcate the boundaries of isoforms based on short-read sequencing, with higher efficiency and lower cost than existing long-read sequencing methods. In conjunction with machine learning algorithms, scRCAT-seq demarcates RNA transcripts with unprecedented accuracy. We identified hundreds of previously uncharacterized transcripts and thousands of alternative transcripts for known genes, revealed cell-type specific isoforms for various cell types across different species, and generated a cell atlas of isoform dynamics during the development of retinal cones.

RNA polymerase II speed: a key player in controlling and adapting transcriptome composition

“…Most eukaryotic promoters are known to utilize several different TSSs; nevertheless, how the TSSs are chosen remains an open question. Transcripts with different start sites can differ in their stability or translational efficiency (Rojas‐Duran & Gilbert, 2012 ) and give rise to transcriptome variations during development and cell differentiation (Hu et al , 2020 ). In budding yeast, RNA Pol II speed controls the use of differential TSSs, with fast RNA Pol II mutants shifting to upstream TSSs, while slow RNA Pol II mutants shift to downstream TSSs (Kaplan et al , 2012 ; Braberg et al , 2013 ; Qiu et al , 2020 ).…”

“…CDK12 and CDK13 are multitask RNA Pol II CTD kinases playing important roles for proper gene expression and genome stability (Greenleaf, 2019). Dual inhibition of both kinases significantly decreases RNA Pol II CTD Ser2 and Thr4 phosphorylation, as well as RNA Pol II processivity and speed (Fan et al, 2020).…”

Isoform-specific regulation of rhythmic gene expression by alternative polyadenylation

“…Differences in rhythmic expression between APA isoforms does not appear to rely on a single mechanism, but rather seems to involve several independent processes. While many transcript isoforms are expressed in specific cell subtypes (e.g., 24.4% in mouse liver; Figure 2) (Booeshaghi et al, 2020;Hu et al, 2020;Lianoglou et al, 2013), cell subtypespecificity only contributes moderately to the differential rhythmicity between of APA isoforms. In contrast, posttranscriptional regulation is highly prevalent, especially for the generation of rhythmic APA isoforms from arrhythmically transcribed transcripts (i.e., Group 1 isoforms, Figure 3).…”

Single-cell technologies and analyses in hematopoiesis and hematological malignancies

“…In this respect, a singlecell total RNA-seq method has recently been developed that captures full-transcript fragments of polyadenylated and non-polyadenylated transcripts (with the exception of unwanted ribosomal cDNAs) and analyzes them in an unbiased and strand-specific manner by employing random primers and a template-switching mechanism [27]. Novel strategies to manage the splicing problem include adding a specific sequence tag (containing the UMI and cell barcode) to both ends of full-length cDNAs [28] or combining high-throughput sequencing methods with high-accuracy cell barcoding and UMI assignment [29].…”