A potential role for initiator-tRNA in pre-mRNA splicing regulation

Abstract

The translation initiator-tRNA plays a crucial role in the initiation of protein synthesis in both prokaryotic and eukaryotic cells, by employing specific base pairing between its anticodon triplet CAU and the general initiation codon AUG in the mRNA. Here we show that the initiator-tRNA may also act, in a manner that is independent of its role in protein translation, as a pre-mRNA splicing regulator. Specifically, we show that alternative splicing events that are induced by mutations in the translation initiation AUG codon can be suppressed by expressing initiator-tRNA constructs carrying anticodon mutations that compensate for the AUG mutations. These mutated initiator-tRNAs appeared to be uncharged with an amino acid. Our results imply that recognition of the initiation AUG sequence by the anticodon triplet of initiator-tRNA in its unloaded state plays a role in quality control of splicing in the cell nucleus by a yet unresolved mechanism. Identifying the initiatortRNA as a transacting splicing regulator suggests a novel involvement of this molecule in splicing regulation and provides a critical step toward deciphering this intriguing mechanism.5′ splice site | suppression of splicing | alternative splicing | premature termination codon | spliceosome A key step in both constitutive and alternative pre-mRNA splicing involves the recognition and selection of a consensus sequence that defines the 5′ splice site (5′SS) (1). However, a genomic survey of 446 genes revealed that sequences that comply with the 5′SS consensus, but are not used for splicing (hence termed latent 5′SSs), are highly abundant within introns (2). More than 95% of these latent 5′SSs are preceded by an in-frame stop codon, whose abundance is significantly greater than that expected by chance alone (2, 3). These findings invoked an RNA proofreading mechanism proposed to prevent the use of latent 5′ SSs that otherwise would generate transcripts with premature termination codons (PTCs) (4).The existence of such a putative stop codon-mediated suppression of splicing (SOS) mechanism was first suggested by examining the splicing patterns of transcripts expressed from the constitutive gene encoding the multifunctional protein CAD (carbamoyl-phosphate synthetase, aspartate transcarbamylase, dihydroorotase) in Syrian hamster cells subjected to heat shock stress. Under these conditions, an intronic latent 5′SS, which conformed to the 5′SS consensus better than the upstream authentic one, was activated by the heat shock treatment, whereas under normal growth conditions splicing at that site could not be detected (5). It was also noted that the sequence flanked by the latent 5′SS and the upstream authentic 5′SS contained four stop codons in the CAD protein reading frame. This finding suggested that suppression of splicing at the latent 5′SS could be attributed to the occurrence of these in-frame stop codons. An experimental test of SOS was then carried out using CAD minigene constructs in which all or part of the four in-frame stop codons that reside in ...

Genome-wide activation of latent donor splice sites in stress and disease

“…In previous studies, we have shown that the apparent lack of expression of latent mRNAs under normal growth conditions could not be attributed to degradation by NMD or any other NMD-like RNA surveillance mechanism [(5) and summarized in Supplementary Table S1 of (34)]. Our data have suggested an alternative model, whereby splicing involving latent sites is suppressed such that the generation of PTC-bearing mRNAs is avoided (3,18,31,34).…”

“…Our data have suggested an alternative model, whereby splicing involving latent sites is suppressed such that the generation of PTC-bearing mRNAs is avoided (3,18,31,34). However, we see here that subjecting cells to stresses appeared to have abrogated this suppression, leading to the generation of latent mRNAs.…”

Function of alternative splicing

“…A new cloning method provided evidence for widespread expression of dsRNAs in mouse brain, suggesting that pre-mRNA regulation by antisense RNAs is much more common than previously anticipated (Shen et al, 2011). tRNAs are another emerging regulation by RNA where the initiator tRNA, devoid of a loaded methionine, can regulate alternative splicing events that depend on the AUG start codon for their usage (Kamhi et al, 2010). …”

Apoptotic regulation and tRNA

“…Second, while the translation initiator tRNA i Met plays a crucial role in the initiation of protein synthesis, it also has the ability to act as a pre-mRNA splicing regulator in a manner independent of its role in protein synthesis. Specifically, alternative splicing events resulting from AUG codon mutations are suppressible by initiator tRNA i Met variants harboring anticodon mutations that match the AUG mutations (Kamhi et al, 2010). This mechanism of regulation of splicing appears to play a role in quality control of splicing in the cell nucleus, preventing the generation of premature termination codons.…”