Cis- andtrans-splicing of mRNAs mediated by tRNA sequences in eukaryotic cells

Abstract

The formation of chimeric mRNAs is a strategy used by human cells to increase the complexity of their proteome, as revealed by the ENCODE project. Here, we use Saccharomyces cerevisiae to show a way by which trans-spliced mRNAs can be generated. We demonstrate that a pretRNA inserted into a premRNA context directs the splicing reaction precisely to the sites of the tRNA intron. A suppressor pretRNA gene was inserted, in cis, into the sequence encoding the third cytoplasmic loop of the Ste2 or Ste3 G proteincoupled receptor. The hybrid RNAs are spliced at the specific pretRNA splicing sites, releasing both functional tRNAs that suppress nonsense mutations and translatable mRNAs that activate the signal transduction pathway. The RNA molecules extracted from yeast cells were amplified by RT-PCR, and their sequences were determined, confirming the identity of the splice junctions. We then constructed two fusions between the premRNA sequence (STE2 or STE3) and the 5-or 3-pretRNA half, so that the two hybrid RNAs can associate with each other, in trans, through their tRNA halves. Splicing occurs at the predicted pretRNA sites, producing a chimeric STE3-STE2 receptor mRNA. RNA trans-splicing mediated by tRNA sequences, therefore, is a mechanism capable of producing new kinds of RNAs, which could code for novel proteins.ENCODE ͉ G protein-coupled receptors ͉ genomics ͉ tRNA endonuclease I n higher eukaryotes, most genes contain one or more introns, which are removed by the spliceosomal complex in a regulated manner. When multiple introns are present, alternative splicing provides a way to increase the number of mRNAs from each gene and hence to enhance protein diversity. In lower eukaryotes, like the yeast Saccharomyces cerevisiae, only Ϸ250 genes of Ͼ6,000 contain an intron, and for most of them, a single intron is present. Splice-site sequences in yeast introns are characterized by a strict consensus, and alternative splicing is rare (1, 2).The formation of chimeric mRNAs constitutes another strategy that eukaryotic cells can use to increase proteome complexity. This phenomenon is much more widely spread than previously thought. Recent studies have shown that many of the human genes analyzed in the ENCODE project use exons lying outside their canonical boundaries (3, 4).Chimeric mRNAs can be generated by several mechanisms. In one way, an exon of one gene is fused to an exon of a different gene in a reaction mediated by the spliceosome complex. This fusion can occur in cis, when the two exons derive from two adjacent cotranscribed genes (5, 6), or in trans, when the two exons come from different premRNA transcripts (7,8). Another way for the generation of chimeric transcripts requires the use of a tRNA-splicing endonuclease. We have shown that an endonuclease of archaeal origin can carry out trans-and cis-splicing of mRNA in cultured mouse cells (9).The expression of tRNA genes in most organisms requires the accurate removal of intervening sequences. In Bacteria, pretRNA splicing is autocatalytic, whereas in Archaea a...

Long-distance splicing

“…Importantly, in archaea there is also a precedent for trans-splicing of tRNA halves (19). Thus, the two key requirements for pre-mRNA splicing in trans by the tRNA-splicing machinery, proven in principle by Di Segni et al (5), have already been observed in nature.…”

“…In this issue, Di Segni et al (5) designed an elegant system to ask whether tRNAs can be used to recruit the endogenous tRNA-splicing machinery in yeast to mediate mRNA splicing (5). The authors engineered constructs that would produce hybrid pre-tRNA/pre-mRNAs.…”

Yeast pheromone receptor genes STE2 and STE3 are differently regulated at the transcription and polyadenylation level

“…We used the strain DDS2 (ref. 6), in which the endogenous STE2 gene, as well as the STE3 gene, were previously deleted. Moreover, this strain is devoid of the FAR1 gene, responsible for growth arrest; DDS2, consequently, is able to grow even when the mating pathway is autocrinally activated.…”

tRNA biology charges to the front

“…Another recent example of the flexibility of tRNA processing machinery is the demonstration in yeast that a designed STE2 mRNA containing an embedded introncontaining pre-tRNA can be spliced by the tRNA splicing machinery and RNase P to yield both a functional Ste2 protein and a functional tRNA (Di Segni et al 2008).…”