Identification and characterization of critical cis-acting sequences within the yeast Ty1 retrotransposon

Abstract

The yeast long terminal repeat (LTR) retrotransposon Ty1, like retroviruses, encodes a terminally redundant RNA, which is packaged into virus-like particles (VLPs) and is converted to a DNA copy by the process of reverse transcription. Mutations predicted to interfere with the priming events during reverse transcription and hence inhibit replication are known to dramatically decrease transposition of Ty1. However, additional cis-acting sequences responsible for Ty1 replication and RNA dimerization and packaging have remained elusive. Here we describe a modular mini-Ty1 element encoding the minimal sequence that can be retrotransposed by the Ty1 proteins, supplied in trans by a helper construct. Using a mutagenic screening strategy, we recovered transposition-deficient modular mini-Ty1-HIS3 elements with mutations in sequences required in cis for Ty1 replication and integration. Two distinct clusters of mutations mapped near the 5-end of the Ty1 RNA. The clusters define a GAGGAGA sequence at the extreme 5-end of the Ty1 transcript and a complementary downstream UCUCCUC sequence, 264 nt into the RNA. Disruption of the reverse complementarity of these two sequences decreased transposition and restoration of complementarity rescued transposition to wild-type levels. Ty1 cDNA was reduced in cells expressing RNAs with mutations in either of these short sequences, despite nearly normal levels of Ty1 RNA and VLPs. Our results suggest that the intramolecular interaction between the 5-GAGGAGA and UCUCCUC sequences stabilizes an RNA structure required for efficient initiation of reverse transcription. Keywords: RNA; intramolecular interaction; mutagenesisTy1 is one of five types of long terminal repeat (LTR) retrotransposons inhabiting the genome of the yeast Saccharomyces cerevisiae. LTR retrotransposons, such as Ty1, are similar to retroviruses in many aspects (Boeke and Stoye 1997;Voytas and Boeke 2002). Analogous to retroviruses, Ty1 elements consist of a central coding region flanked by directly repeated LTRs, which are divided into the U3, R, and U5 regions. The Ty1 lifecycle, a streamlined version of the retroviral lifecycle, can be divided into three phases: (1) expression and assembly, (2) reverse transcription, and (3) integration. Transcription of Ty1 produces a terminally redundant RNA molecule, which serves dual functions. First, translation of the element-encoded RNA generates the structural protein Gag and enzyme Pol, which are required for transposition, specifically for replication and integration of Ty1. Pol is made as a Gag-Pol read-through protein by translational frameshifting (Belcourt and Farabaugh 1990). Gag and Gag-Pol proteins are coassembled with Ty1 RNA and the yeast initiator tRNA iMet within the cytoplasm, forming virus-like particles (VLPs) (Chapman et al. 1992;Roth 2000;Voytas and Boeke 2002). VLPs are direct transposition intermediates in which reverse transcription occurs Eichinger and Boeke 1988). Second, during Ty1 replication, as in retroviruses, the terminally redundant RNA, th...

Posttranslational interference of Ty1 retrotransposition by antisense RNAs

“…5). Alternatively, Ty1 mRNA/AS RNA duplexes may inhibit Ty1 mRNA dimer formation or other mRNA interactions (3,29), or prevent annealing of tRNA-Met i with the pbs. As a result, Ty1AS RNA-mediated (Fig.…”

“…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.…”

Retrotransposon Ty1 RNA contains a 5′-terminal long-range pseudoknot required for efficient reverse transcription

“…Among other roles, this region sequesters the cellular tRNA i-Met , which serves as the primer of reverse transcription, and produces a short DNA molecule referred to as minus strand strong stop DNA (Chapman et al 1992;Wilhelm et al 1994;Keeney et al 1995). Previous studies have predicted a variety of structures for this RNA based on modeling, phylogenetic analyses, and mutational studies (Friant et al 1996(Friant et al , 1997(Friant et al , 1998Cristofari et al 2002;Bolton et al 2005).…”

Novel Transcript Truncating Function of Rap1p Revealed by Synthetic Codon-Optimized Ty1 Retrotransposon

“…Not only does this "mRNA" code for the GAG and POL proteins required for retrotransposition, but it also serves a critical function as the genetic material for replication. Both Ty1 mRNA and its cDNA product contain cis-acting nucleotide determinants required for retrotransposition.Mutagenesis of Ty1 and screening for cis-acting nucleotide determinants has been previously performed with various strategies, including in-frame linker insertion mutagenesis Devine and Boeke 1994;Monokian et al 1994), analysis of synthetic Ty1 DNA fragments to determine minimal requirements for the integration reaction (Eichinger and Boeke 1990;Braiterman et al 1994;Devine and Boeke 1994;Sharon et al 1994), deletion analysis and PCR-mediated mutagenesis of mini-Ty1 elements (Xu and Boeke 1990;Bolton et al 2005), and comparative sequence analysis followed by targeted mutagenesis and/or generation of complementary mutations in RNA stem regions (Chapman et al 1992;Heyman et al 1995;Lauermann et al 1995;Friant et al 1998;Cristofari et al 2002;Bolton et al 2005). These studies provided insights into mechanisms underlying Ty1 retrotransposition and revealed several cis-acting sequences, including the Met i -tRNA:primer binding site (PBS) interaction (Chapman et al 1992; Friant et al 1998), LTRs (Eichinger andBraiterman et al 1994;Devine and Boeke 1994;Sharon et al 1994), polypurine tracts (PPTs) (Heyman et al 1995;Lauermann et al 1995), the GAG-POL frameshift region (Belcourt and Farabaugh 1990; Lawler et al 2001), CYC5 and CYC3 (Cristofari et al 2002), and an extensive 59 RNA structure required for efficient initiation of reverse transcription (Bolton et al 2005).…”

“…These studies provided insights into mechanisms underlying Ty1 retrotransposition and revealed several cis-acting sequences, including the Met i -tRNA:primer binding site (PBS) interaction (Chapman et al 1992; Friant et al 1998), LTRs (Eichinger andBraiterman et al 1994;Devine and Boeke 1994;Sharon et al 1994), polypurine tracts (PPTs) (Heyman et al 1995;Lauermann et al 1995), the GAG-POL frameshift region (Belcourt and Farabaugh 1990; Lawler et al 2001), CYC5 and CYC3 (Cristofari et al 2002), and an extensive 59 RNA structure required for efficient initiation of reverse transcription (Bolton et al 2005). Additional cis-sequences may remain undiscovered in Ty1.…”