Depletion of REF/Aly alters gene expression and reduces RNA polymerase II occupancy

Abstract

Pre-mRNA processing is mechanistically linked to transcription with RNA pol II serving as a platform to recruit RNA processing factors to nascent transcripts. The TREX complex member, REF/Aly, has been suggested to play roles in transcription and nuclear RNA stability in addition to its more broadly characterized role in mRNA export. We employed RNA-seq to identify a subset of transcripts with decreased expression in both nuclear and cytoplasmic fractions upon REF/Aly knockdown, which implies that REF/Aly affects their expression upstream of its role in mRNA export. Transcription inhibition experiments and metabolic labeling assays argue that REF/Aly does not affect stability of selected candidate transcripts. Instead, ChIP assays and nuclear run-on analysis reveal that REF/Aly depletion diminishes the transcription of these candidate genes. Furthermore, we determined that REF/Aly binds directly to candidate transcripts, supporting a direct effect of REF/Aly on candidate gene transcription. Taken together, our data suggest that the importance of REF/Aly is not limited to RNA export, but that REF/Aly is also critical for gene expression at the level of transcription. Our data are consistent with the model that REF/Aly is involved in linking splicing with transcription efficiency.

Pharmacological inhibition of the spliceosome subunit SF3b triggers exon junction complex-independent nonsense-mediated decay

“…6E). This could be due to less-efficient transcription upon ALYREF depletion, as recently reported (Stubbs and Conrad, 2015).…”

The role of TREX in gene expression and disease

“…Like DDX39A/B, ALYREF is displaced from the mRNP before nucleocytoplasmic translocation [61,68]. ALYREF is involved in the export of both spliced and intronless mRNAs [69], and RNA interference (RNAi)-mediated knockdown of ALYREF leads to disruption of mRNA export for around 3900 mRNAs in 293T cells [70]. Functional redundancy between ALYREF and other mRNA export factors such as UIF (described below) may account for the relatively small number of mRNAs whose export is affected following ALYREF RNAi.…”

“…It appears that this is also the case for transcription and export because TREX is required for efficient transcription elongation, particularly of long genes [21,87]. ALYREF affects transcription of a subset of genes [70], suggesting a reciprocal coupling between transcription and export via ALYREF, as it has been observed for other steps in the gene expression pathway [88]. …”

ITRAQ-Based Proteomics Analysis of Triptolide On Human A549 Lung Adenocarcinoma Cells

“…REF1/Aly is a nuclear speckle protein implicated in mRNA export and is a physiological target of the nuclear Akt signaling cascade [42,43]. The depletion of Aly markedly blocks cell cycle progression and reduces cell growth and mRNA export, and these processes are regulated by Akt phosphorylation [43,44]. GTPBP4, located in the nucleus, is involved in the biosynthesis of the 60S subunit of the ribosome [45] and can be used as a molecular switch to control signal transduction pathways, protein synthesis and other biological processes.…”