AlternativeRESTSplicing Underappreciated

Abstract

As a major orchestrator of the cellular epigenome, the repressor element-1 silencing transcription factor (REST) can either repress or activate thousands of genes depending on cellular context, suggesting a highly context-dependent REST function tuned by environmental cues. While REST shows cell-type non-selective active transcription 1 , an N-terminal REST4 isoform caused by alternative splicing -inclusion of an extra exon (N 3c ) which introduces a premature stop codon -has been implicated in neurogenesis and tumorigenesis [2][3][4][5] . Recently, in line with established epigenetic regulation of pre-mRNA splicing 6,7 , we demonstrated that REST undergoes extensive, context-dependent alternative splicing which results in the formation of a large number of mRNA variants predictive of multiple protein isoforms 8 . Supported by that immunoblotting/-staining with different anti-REST antibodies yield inconsistent results, alternative splicing allows production of various structurally and functionally different REST protein isoforms in response to shifting physiological requirements, providing a reasonable explanation for the diverse, highly context-dependent REST function. However, REST isoforms might be differentially assayed or manipulated, leading to data misinterpretation and controversial findings. For example, in contrast to the proposed neurotoxicity of elevated nuclear REST in ischemia 9 and Huntington's disease 10,11 , Lu et al. recently reported decreased nuclear REST in Alzheimer's disease and neuroprotection of REST in ageing brain 12 . Unfortunately, alternative REST splicing was largely neglected by Lu et al., making it necessary for a reevaluation of their findings.As shown in Fig.1a, human REST gene boundary is now doubled by an alternative last exon (E 5 ) which is mutually exclusive to E 4 . While numerous novel alternative exons and 5'/3' ends were identified, the 3 constitutive exons (E 2 , E 3 and E 4 ) comprising the open reading frame (ORF) of REST can be skipped partially or completely, alone or in combination, producing at least 45 mRNA variants predictive of multiple protein isoforms (Fig.1b) 8 . For example, REST4 -which was first described in rat as a group of REST isoforms 4 , is predicted by multiple mRNA variants (e.g. JX896958, JX896971 and JX896983) with E 3 followed by variable exons introducing a premature stop codon, suggesting that like the case in rat, human REST4 is also a group of isoforms, but not a single mRNA/protein isoform. Meanwhile, REST1 -another N-terminal isoform is predicted by multiple mRNA variants lacking E 3 . Notably, for E 2 -skipped variants (e.g. XM_005265760 and JX896960) missing the conventional start codon, an in-frame AUG in E 3 may initiate translation of a C-terminal REST C isoform (XP_005265817), which was recently described in Rest conditional knockout (cKO) mice 13 . In addition, some mRNA variants (e.g. JX896978 and KC117266) with partial E 2 skipping are predictive of proteins missing variable . CC-BY-ND 4.0 International license peer-review...

The Genome-Wide Binding Profile for Human RE1 Silencing Transcription Factor Unveils a Unique Genetic Circuitry in Hippocampus

“…1A,B). Of note, this REST antibody will not detect truncated splice variants that are still largely enigmatic in terms of their function (Chen and Miller, 2018).…”

Modulation of nuclear REST by alternative splicing: a potential therapeutic target for Huntington's disease

“…In the study of Lu et al . , only the full‐length REST was actually considered; however, qRT‐PCR with four primer sets targeting different exons of REST yielded different expression levels, providing evidence for the existence of alternative REST splicing . Notably, Lu et al .…”

“…Notably, Lu et al . performed immunostaining with multiple anti‐REST antibodies without considering differences between the antibodies and disclosing detailed usage of the antibodies, making it possible that nuclear REST differences between the experimental groups might be confounded by biased usage of the antibodies for samples of different groups .…”

Genome-wide assessment of REST binding profiles reveals distinctions between human and mouse hippocampus

“…Western blot immuno-labeling and qChIP in Rest knockout mice confirmed specificity of the antibody to full length REST protein [15] (Additional file 1: Figure S1.1d-e). Of note, this antibody will not recognize truncated splice variants [32], whose functional importance and expression levels are still characterized poorly and are not considered in this study.…”