Splicing promotes the nuclear export of β-globin mRNA by overcoming nuclear retention elements

Abstract

Most current models of mRNA nuclear export in vertebrate cells assume that an mRNA must have specialized signals in order to be exported from the nucleus. Under such a scenario, mRNAs that lack these specialized signals would be shunted into a default pathway where they are retained in the nucleus and eventually degraded. These ideas were based on the selective use of model mRNA reporters. For example, it has been shown that splicing promotes the nuclear export of certain model mRNAs, such as human β-globin, and that in the absence of splicing, the cDNA-derived mRNA is retained in the nucleus and degraded. Here we provide evidence that β-globin mRNA contains an element that actively retains it in the nucleus and degrades it. Interestingly, this nuclear retention activity can be overcome by increasing the length of the mRNA or by splicing. Our results suggest that contrary to many current models, the default pathway for most intronless RNAs is to be exported from the nucleus, unless the RNA contains elements that actively promote its nuclear retention.

TPR is required for the nuclear export of mRNAs and lncRNAs from intronless and intron-poor genes

“…All ftz and βG reporter construct in pcDNA3.0 were described as previously (24)(25)(26)29). To generate the USP14-3'UTR containing plasmids, we amplified a portion of the USP14 3'UTR from U2OS genomic DNA using the forward primer: 5'-CTCGAGATTA AGTTTGATGA TGACAAAGTC A-3' and reverse primer: 5'-TCTAGACAGT CAGAAAATCT AAATTCTAAT TGTT-3'.…”

“…The lentiviral delivered shRNA protein depletion is as previously described (24,25,58). Briefly, HEK293T were plated at 50% confluency on 60 mm dishes and transiently transfected with the gene specific shRNA pLKO.1 plasmid (Sigma), packaging plasmid (Δ8.9) and envelope (VSVG) vectors using Lipo 293T DNA in vitro transfection reagent (SignaGen Laboratories) according to the manufacture's protocol.…”

“…Indeed splicing has been shown to recruit certain TREX components, such as UAP56, to the messenger ribonucleoprotein (mRNP) complex (2,20,21). Nonetheless, TREX components have also been shown to be efficiently loaded onto mRNAs even in the absence of splicing (22)(23)(24)(25)(26). TREX may be recruited by RNA polymerase II in a co-transcriptional manner (1), by the nuclear cap-binding complex (27) and by the 3' cleavage and polyadenylation machinery (28).…”

“…As such, TREX components are required for the export of non-spliced mRNAs (22,23,29). In particular cases where splicing enhances nuclear export, it does so by overriding the activity nuclear retention signals (25), although the exact mechanism for how it does so remains unclear. In addition, it has been reported that additional factors are required for the export of naturally intronless mRNAs (30,31), but to date the role of these factors has not been investigated on a transcriptome-wide scale.…”

Ribosome biogenesis is a downstream effector of the oncogenic U2AF1-S34F mutation

“…Total RNA was extracted using Trizol (Thermo) according to the manufacturer's instructions. The RNA was separated on a denaturing gel and transferred to nylon membrane (GE healthcare) overnight as previously described (Akef et al, 2015). The membrane was hybridized to a 32 Plabeled probe overnight and imaged using a Typhoon phosphoimager (Molecular Dynamics).…”