A role for TREX components in the release of spliced mRNA from nuclear speckle domains

Abstract

The TREX complex, which functions in mRNA export, is recruited to mRNA during splicing. Both the splicing machinery and the TREX complex are concentrated in 20–50 discrete foci known as nuclear speckle domains. Using a model system where CMV-DNA constructs were microinjected into HeLa cell nuclei, we have followed the fates of the transcripts. Here we show that transcripts lacking functional splice sites, which are inefficiently exported, do not associate with nuclear speckle domains but are instead distributed throughout the nucleoplasm. In contrast, pre-mRNAs containing functional splice sites accumulate in nuclear speckles, and our data suggest that splicing occurs in these domains. When the TREX components UAP56 or Aly are knocked down, spliced mRNA, as well as total polyA+ RNA, accumulates in nuclear speckle domains. Together, our data raise the possibility that pre-mRNA undergoes splicing in nuclear speckle domains, before release by TREX components for efficient export to the cytoplasm.

Post-transcriptional spliceosomes are retained in nuclear speckles until splicing completion

“…However, we cannot exclude that post-transcriptional splicing may additionally or alternatively occur elsewhere in the nucleus. That speckles might serve not only as storage sites for splicing factors but also as sites for splicing per se, was suggested by previous studies 23,24,41,42 . Retention of spliceosomes (and their pre-mRNA substrate) in speckles could be due to the fact that all splicing factors are thought to possess their own speckle localization signal; thus spliceosomes, which are composed of a multitude of splicing factors, might have a high affinity for speckles 20 .…”

“…Indeed, previous analyses of pre-mRNA splicing in mammalian cells were limited to individual pre-mRNA species or small subsets of pre-mRNAs, and were followed in situ by FISH, ChIP or, after nuclear fractionation, by RT-PCR 7,9,39 . The fate of a handful of pre-mRNAs was followed by microinjection of a reporter gene or transcript 23,[40][41][42] , which might not reflect the same fate as that of nascent transcripts transcribed from chromatinassociated genes. Thus, an added advantage of our antibodies is that they allow us to follow the fate of pre-mRNAs that are transcribed and spliced under physiological conditions.…”

“…We next tested whether release of poly(A) + RNA from speckles induced by CDC5L complementation is coupled to its export to the cytoplasm, by additionally blocking mRNA export via knockdown of the export essential factor Aly 23 . RNAi-mediated knockdown of both CDC5L and Aly led to a ~75% and ~90% reduction in each protein, respectively, after 48 h (Fig.…”

“…Nuclear speckles are believed to be storage sites for splicing factors 20 , and are surrounded by perichromatin fibrils where splicing is thought to occur 21,22 . Recent FISH studies suggest that splicing might occur directly in speckles 23 . However, as FISH does not allow the direct localization of active spliceosomes, and the RNAs followed in these studies were not generated from genes packaged in chromatin, additional data are still required to clearly elucidate the subnuclear location of co-and post-transcriptional splicing.…”

The lncRNA MIR31HG regulates p16INK4A expression to modulate senescence

“…It has been recently reported that a subset of lncRNAs are exported to the cytoplasm via common mRNA pathways 39 . To corroborate the export of MIR31HG during senescence, we depleted a set of export factors that have been previously involved in RNA export 40,41 ( Supplementary Fig. 5f).…”