An intron with a constitutive transport element is retained in a Tap messenger RNA

Abstract

Alternative splicing is a key factor contributing to genetic diversity and evolution. Intron retention, one form of alternative splicing, is common in plants but rare in higher eukaryotes, because messenger RNAs with retained introns are subject to cellular restriction at the level of cytoplasmic export and expression. Often, retention of internal introns restricts the export of these mRNAs and makes them the targets for degradation by the cellular nonsense-mediated decay machinery if they contain premature stop codons. In fact, many of the database entries for complementary DNAs with retained introns represent them as artefacts that would not affect the proteome. Retroviruses are important model systems in studies of regulation of RNAs with retained introns, because their genomic and mRNAs contain one or more unspliced introns. For example, Mason-Pfizer monkey virus overcomes cellular restrictions by using a cis-acting RNA element known as the constitutive transport element (CTE). The CTE interacts directly with the Tap protein (also known as nuclear RNA export factor 1, encoded by NXF1), which is thought to be a principal export receptor for cellular mRNA, leading to the hypothesis that cellular mRNAs with retained introns use cellular CTE equivalents to overcome restrictions to their expression. Here we show that the Tap gene contains a functional CTE in its alternatively spliced intron 10. Tap mRNA containing this intron is exported to the cytoplasm and is present in polyribosomes. A small Tap protein is encoded by this mRNA and can be detected in human and monkey cells. Our results indicate that Tap regulates expression of its own intron-containing RNA through a CTE-mediated mechanism. Thus, CTEs are likely to be important elements that facilitate efficient expression of mammalian mRNAs with retained introns.

Mutually exclusive interactions drive handover of mRNA from export adaptors to TAP

“…When TAP is tethered to pre-mRNA or over expressed it stimulates the direct export of pre-mRNA (23). Simple retroviruses and TAP pre-mRNA have taken advantage of this property by evolving specific high-affinity RNA elements that directly bind TAP, recruiting it and promoting export of RNAs that would normally be exported inefficiently (29,30). Were TAP to directly bind all cellular pre-mRNAs efficiently, it might lead to premature export, a disastrous situation for the cell, so it is likely that this process is regulated.…”

Fragile X mental retardation protein FMRP and the RNA export factor NXF2 associate with and destabilize Nxf1 mRNA in neuronal cells

“…Although there have been extensive studies on the function of NXF1, much less is known about the regulation of its expression. However, the Hammarskjöld group (28) has recently demonstrated that there exists at least one alternatively spliced Nxf1 variant that encodes a truncated form of NXF1 and that the expression of this variant is regulated by the full-length NXF1. Our findings represent yet another mechanism of Nxf1 regulation.…”

Involvement of the RNA-binding protein ARE/poly(U)-binding factor 1 (AUF1) in the cytotoxic effects of proinflammatory cytokines on pancreatic beta cells

“…Subcellular fractionation and protein extraction Subcellular fractionation was performed as described by Li et al [19]. Briefly, cells were lysed for 15 min on ice using a Triton X-100 lysis buffer (50 mmol/l Tris/HCl, pH 7.5, 0.5% Triton X-100, 137.5 mmol/l NaCl, 10% glycerol, 1 mmol/l sodium vanadate, 50 mmol/l NaF, 10 mmol/l sodium pyrophosphate, 5 mmol/l EDTA and the Protease Inhibitors Cocktail [Sigma, St Louis, MO, USA]).…”