Cis- and trans-splicing of mRNAs mediated by tRNA sequences in eukaryotic cells.
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.
Di Segni
Gianfranco
G
Istituto di Biologia Cellulare, Consiglio Nazionale delle Ricerche, Via Ramarini 32, 00015 Monterotondo (Rome), Italy.
Gastaldi
Serena
S
Tocchini-Valentini
Glauco P
GP
D001483:Base Sequence; D005057:Eukaryotic Cells / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D049770:Genes, Mating Type, Fungal; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes; D014176:Protein Biosynthesis; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics; D022821:RNA Splice Sites / Q000235:genetics; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D012343:RNA, Transfer / Q000737:chemistry / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000235:genetics*; D017384:Sequence Deletion; D015398:Signal Transduction; D013489:Suppression, Genetic; D020040:Trans-Splicing / Q000235:genetics*
D009692:Nucleic Acid Heteroduplexes; D012322:RNA Precursors; D022821:RNA Splice Sites; D012333:RNA, Messenger; D012343:RNA, Transfer