Inhibition of U4 snRNA in Human Cells Causes the Stable Retention of Polyadenylated Pre-mRNA in the Nucleus

Abstract

Most human pre-mRNAs contain introns that are removed by splicing. Such a complex process needs strict control and regulation in order to prevent the expression of aberrant or unprocessed transcripts. To analyse the fate of pre-mRNAs that cannot be spliced, we inhibited splicing using an anti-sense morpholino (AMO) against U4 snRNA. As a consequence, splicing of several selected transcripts was strongly inhibited. This was accompanied by the formation of enlarged nuclear speckles containing polyadenylated RNA, splicing factors and the nuclear poly(A) binding protein. Consistently, more polyadenylated pre-mRNA could be isolated from nucleoplasmic as well as chromatin-associated RNA fractions following U4 inhibition. Further analysis demonstrated that accumulated pre-mRNAs were stable in the nucleus and that nuclear RNA degradation factors did not re-localise to nuclear speckles following splicing inhibition. The accumulation of pre-mRNA and the formation of enlarged speckles were sensitive to depletion of the 3′ end processing factor, CPSF73, suggesting a requirement for poly(A) site processing in this mechanism. Finally, we provide evidence that the pre-mRNAs produced following U4 snRNA inhibition remain competent for splicing, perhaps providing a biological explanation for their stability. These data further characterise processes ensuring the nuclear retention of pre-mRNA that cannot be spliced and suggest that, in some cases, unspliced transcripts can complete splicing sometime after their initial synthesis.

p38MAPK/MK2-mediated phosphorylation of RBM7 regulates the human nuclear exosome targeting complex

“…S2A). As reported for human RBM7 (Lubas et al 2011;Hett and West 2014), these proteins were exclusively localized to the nuclear compartment and did not display notable changes in localization upon anisomycin-treatment. Established "bona fide" substrates of MK2, such as Hsp27 (Stokoe et al 1992) and NOGO-B (Rousseau et al 2005), displayed similar kinetics of phosphorylation (Fig.…”

Characterisation of the biflavonoid hinokiflavone as a pre-mRNA splicing modulator that inhibits SENP

“…Previous studies showed that treatment of cells with either SF3B1 inhibitors, such as meayamycin, spliceostatin, or platinolide B, or the inhibition of U4 snRNA, each lead to the accumulation of polyadenylated RNA in enlarged splicing speckles ( Hett and West, 2014 ; Kaida et al, 2007 ). We therefore investigated whether the mega speckles induced by hinokiflavone also contained polyadenylated RNA.…”

Nusinersen ameliorates motor function and prevents motoneuron Cajal body disassembly and abnormal poly(A) RNA distribution in a SMA mouse model

“…Moreover, nuclear retention of polyadenylated mRNAs may be linked to defective snRNP biogenesis in CBs. In this sense, a recent study 67 reported that the inhibition of U4 snRNP biogenesis, a key component for U4/U6-U5 tri-snRNP assembly in CBs 68 , results in the abnormal nuclear accumulation of polyadenylated mRNAs. Importantly, the ICV administration of nusinersen in SMN∆7 mice substantially reduced the formation of PARGs and restored the nuclear and cytoplasmic compartmentalization of polyadenylated mRNAs in αMNs.…”