Posttranslational interference of Ty1 retrotransposition by antisense RNAs

Abstract

Transposable elements impact genome function by altering gene expression and causing chromosome rearrangements. As a result, organisms have evolved mechanisms, such as RNA-interference, to minimize the level of transposition. However, organisms without the conserved RNAi pathways, like Saccharomyces cerevisiae, must use other mechanisms to prevent transposon movement. Here, we provide evidence that antisense (AS) RNAs from the retrovirus-like element Ty1 inhibit retrotransposition posttranslationally in Saccharomyces. Multiple Ty1AS transcripts overlap Ty1 sequences necessary for copy number control (CNC) and inhibit transposition in trans. Altering Ty1 copy number or deleting sequences in the CNC region that are required for reverse transcription affect Ty1AS RNA level and Ty1 movement. Ty1AS RNAs are enriched in viruslike particles, and are associated with a dramatic decrease in the level of integrase, less reverse transcriptase, and an inability to synthesize Ty1 cDNA. Thus, Ty1AS RNAs are part of an intrinsic mechanism that limits retrotransposition by reducing the level of proteins required for replication and integration.retrotransposon ͉ virus-like particles ͉ RNA interference ͉ Saccharomyces R etrotransposons replicate through an RNA intermediate and can comprise over half the genome in many eukaryotes. To maintain genome stability, forms of RNAi have evolved to inhibit transposition (1). However, Saccharomyces cerevisiae lacks the genes required for RNAi, yet still maintains control over Ty1 retrotransposition. Ty1 elements and retroviruses have similar structures and strategies for gene expression, and encode functionally equivalent Gag and Pol proteins protease (PR), integrase (IN), and reverse transcriptase (RT) (2). Ty1 mRNA has unique subterminal 5Ј (U5) and 3Ј (U3) sequence motifs and a terminally repetitious (R) region. These sequences are required for reverse transcription and integration and form the U3-R-U5 segments of the LTR that bracket the element in the genome. Adjacent to U5 is the primer-binding site (pbs), where tRNA-Met i anneals with Ty1 mRNA to initiate reverse transcription within cytoplasmic virus-like particles (VLPs). Although not completely defined, sequences within GAG also enhance Ty1 mRNA dimer formation and packaging into VLPs (3, 4). The primary translation products are Gag-p49 and Gag-Pol-p199 that are cleaved by PR to form mature Gag-p45, PR-p20, IN-p71, and RT-p63. An association between IN and RT is required for reverse transcription in vivo and RT activity in vitro (5-7). Retrotransposition is completed by IN-mediated integration of Ty1 cDNA into the genome.Despite high levels of Ty1 mRNA and many functional elements in the genome, transposition occurs at a low rate (8-10). Cells also contain a low level of mature Ty1 proteins (11), and few VLPs are present (12). When an active Ty1 element is fused to the GAL1 promoter and carried on a multicopy pGTy1 plasmid (13), galactose induction overcomes the barriers to transposition. However, the mechanism underlying this obse...

T-body formation precedes virus-like particle maturation inS. cerevisiae

“…We chose this background due to the strong inhibition of Ty1 transposition in the absence of the Xrn1p exonuclease, 17 probably due to the accumulation of Ty1 antisense RNAs that inhibit VLP maturation. 17,18 Consistent with a previous report in reference 2, we observed significantly decreased T-body counts in xrn1Δ cells transformed with an empty vector (Fig. 4).…”

P-Body Components Are Required for Ty1 Retrotransposition during Assembly of Retrotransposition-Competent Virus-Like Particles

“…Fifth, Kem1p may be involved in degrading and trafficking Ty1 antisense RNAs, which may further help prevent their colocalization with Ty1 mRNA. Ty1 antisense RNAs associate with VLPs, where they also interfere with accumulation of RT and IN (43). Therefore, Kem1p may enhance the assembly of functional Ty1 VLPs by segregating mRNA/Gag foci from the antisense transcripts, which restrict retrotransposition.…”

“…Since Lsm1p, Pat1p, and Kem1p are required for the formation of mRNA/Gag foci, we determined if deletion of P-body components affected the localization of Ty1 antisense RNAs by using a DIG-labeled probe corresponding to the 5Ј end of GAG. A probe from this region detects multiple antisense RNAs (43). In ϳ95% of the wild-type cells, antisense RNAs localized in puncta predominantly in the cytoplasm (Fig.…”

“…These antisense RNAs have been reported to decrease retrotransposition by repressing transcription (7) or inhibiting accumulation of RT and IN (43). Antisense RNA degradation is regulated by cellular RNA surveillance pathways, which include the P-bodies.…”

Is the Fungus Magnaporthe Losing DNA Methylation?

“…The transcript of the Ty1 LTR-retrotransposon is known to be very abundant (0.1-0.8% of total RNA) in the cell; however, Ty1 transposition seldom occurs (Elder et al 1981;Curcio et al 1990). Thus, as suggested previously, there is likely to be an effective post-transcriptional defense against TE transposition into the genome at least in certain organisms including M. oryzae (Garfinkel et al 2003;Murata et al 2007;Matsuda and Garfinkel 2009). …”