BIOCHEMISTRY
Cis- and trans-splicing of mRNAs mediated by tRNA sequences in eukaryotic cells
Istituto di Biologia Cellulare, Consiglio Nazionale delle Ricerche, Via Ramarini 32, 00015 Monterotondo (Rome), Italy
Edited by Robert Haselkorn, University of Chicago, Chicago, IL, and approved March 11, 2008 (received for review January 15, 2008)
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 protein-coupled 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
Author contributions: G.D.S. and G.P.T.-V. designed research; S.G. performed research; G.D.S. and S.G. analyzed data; and G.D.S. and G.P.T.-V. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
*To whom correspondence should be addressed. E-mail: gtocchini{at}ibc.cnr.it
© 2008 by The National Academy of Sciences of the USA
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