Local features determine Ty3 targeting frequency at RNA polymerase III transcription start sites.

Retroelement integration into host genomes affects chromosome structure and function. A goal of a considerable number of investigations is to elucidate features influencing insertion site selection. The Saccharomyces cerevisiae Ty3 retrotransposon inserts proximal to the transcription start sites (TSS) of genes transcribed by RNA polymerase III (RNAP3). In this study, differential patterns of insertion were profiled genome-wide using a random barcode-tagged Ty3. Saturation transposition showed that tRNA genes (tDNAs) are targeted at widely different frequencies even within isoacceptor families. Ectopic expression of Ty3 integrase (IN) showed that it localized to targets independent of other Ty3 proteins and cDNA. IN, RNAP3, and transcription factor Brf1 were enriched at tDNA targets with high frequencies of transposition. To examine potential effects of cis-acting DNA features on transposition, targeting was tested on high-copy plasmids with restricted amounts of 5' flanking sequence plus tDNA. Relative activity of targets was reconstituted in these constructions. Weighting of genomic insertions according to frequency identified an A/T-rich sequence followed by C as the dominant site of strand transfer. This site lies immediately adjacent to the adenines previously implicated in the RNAP3 TSS motif (CAA). In silico DNA structural analysis upstream of this motif showed that targets with elevated DNA curvature coincide with reduced integration. We propose that integration mediated by the Ty3 intasome complex (IN and cDNA) is subject to inputs from a combination of host factor occupancy and insertion site architecture, and that this results in the wide range of Ty3 targeting frequencies.

Control of yeast retrotransposons mediated through nucleoporin evolution.

Yeasts serve as hosts to several types of genetic parasites. Few studies have addressed the evolutionary trajectory of yeast genes that control the stable co-existence of these parasites with their host cell. In Saccharomyces yeasts, the retrovirus-like Ty retrotransposons must access the nucleus. We show that several genes encoding components of the yeast nuclear pore complex have experienced natural selection for substitutions that change the encoded protein sequence. By replacing these S. cerevisiae genes with orthologs from other Saccharomyces species, we discovered that natural sequence changes have affected the mobility of Ty retrotransposons. Specifically, changing the genetic sequence of NUP84 or NUP82 to match that of other Saccharomyces species alters the mobility of S. cerevisiae Ty1 and Ty3. Importantly, all tested housekeeping functions of NUP84 and NUP82 remained equivalent across species. Signatures of natural selection, resulting in altered interactions with viruses and parasitic genetic elements, are common in host defense proteins. Yet, few instances have been documented in essential housekeeping proteins. The nuclear pore complex is the gatekeeper of the nucleus. This study shows how the evolution of this large, ubiquitous eukaryotic complex can alter the replication of a molecular parasite, but concurrently maintain essential host functionalities regarding nucleocytoplasmic trafficking.

Function of a retrotransposon nucleocapsid protein.

Long terminal repeat (LTR) retrotransposons are not only the ancient predecessors of retroviruses, but they constitute significant fractions of the genomes of many eukaryotic species. Studies of their structure and function are motivated by opportunities to gain insight into common functions of retroviruses and retrotransposons, diverse mechanisms of intracellular genomic mobility, and host factors that diminish or enhance retrotransposition. This review focuses on the nucleocapsid (NC) protein of a Saccharomyces cerevisiae LTR retrotransposon, the metavirus, Ty3. Retrovirus NC promotes genomic (g)RNA dimerization and packaging, tRNA primer annealing, reverse transcription strand transfers, and host protein interactions with gRNA. Studies of Ty3 NC have revealed key roles for Ty3 NC in formation of retroelement assembly sites (retrosomes), and in chaperoning primer tRNA to both dimerize and circularize Ty3 gRNA. We speculate that Ty3 NC, together with P-body and stress-granule proteins, plays a role in transitioning Ty3 RNA from translation template to gRNA, and that interactions between the acidic spacer domain of Ty3 Gag3 and the adjacent basic NC domain control condensation of the virus-like particle.

Ty3 requires yeast La homologous protein for wild-type frequencies of transposition.

The Saccharomyces cerevisiae retrovirus-like element Ty3 inserts specifically into the initiation sites of genes transcribed by RNA polymerase III (pol III). A strain with a disruption of LHP1, which encodes the homologue of autoantigen La protein, was recovered in a screen for mutants defective in Ty3 transposition. Transposition into a target composed of divergent tRNA genes was decreased eightfold. In lhp1 mutants, Ty3 polyproteins were produced at wild-type levels, assembled into virus-like particles (VLPs) and processed efficiently. The amount of cDNA associated with these particles was about half the amount in a wild-type control at early times, but approached the wild-type level after 48 h of induction. Ty3 integration was examined at two genomic tRNA gene families and two plasmid-borne tRNA promoters. Integration was significantly decreased at one of the tRNA gene families, but was only slightly decreased at the second tRNA gene family. These findings suggest that Lhp1p contributes to Ty3 cDNA synthesis, but might also act at a target-specific step, such as integration.

Mutational analysis of the transcription factor IIIB-DNA target of Ty3 retroelement integration.

The Ty3 retrovirus-like element inserts preferentially at the transcription initiation sites of genes transcribed by RNA polymerase III. The requirements for transcription factor (TF) IIIC and TFIIIB in Ty3 integration into the two initiation sites of the U6 gene carried on pU6LboxB were previously examined. Ty3 integrates at low but detectable frequencies in the presence of TFIIIB subunits Brf1 and TATA-binding protein. Integration increases in the presence of the third subunit, Bdp1. TFIIIC is not essential, but the presence of TFIIIC specifies an orientation of TFIIIB for transcriptional initiation and directs integration to the U6 gene-proximal initiation site. In the current study, recombinant wild type TATA-binding protein, wild type and mutant Brf1, and Bdp1 proteins and highly purified TFIIIC were used to investigate the roles of specific protein domains in Ty3 integration. The amino-terminal half of Brf1, which contains a TFIIB-like repeat, contributed more strongly than the carboxyl-terminal half of Brf1 to Ty3 targeting. Each half of Bdp1 split at amino acid 352 enhanced integration. In the presence of TFIIIB and TFIIIC, the pattern of integration extended downstream by several base pairs compared with the pattern observed in vitro in the absence of TFIIIC and in vivo, suggesting that TFIIIC may not be present on genes targeted by Ty3 in vivo. Mutations in Bdp1 that affect its interaction with TFIIIC resulted in TFIIIC-independent patterns of Ty3 integration. Brf1 zinc ribbon and Bdp1 internal deletion mutants that are competent for polymerase III recruitment but defective in promoter opening were competent for Ty3 integration irrespective of the state of DNA supercoiling. These results extend the similarities between the TFIIIB domains required for transcription and Ty3 integration and also reveal requirements that are specific to transcription.

Ty3 integrase is required for initiation of reverse transcription.

The integrase (IN) encoded by the Saccharomyces cerevisiae retrovirus-like element Ty3 has features found in retrovirus IN proteins including the catalytic triad, an amino-terminal zinc-binding motif, and a nuclear localization sequence. Mutations in the amino- and carboxyl-terminal domains of Ty3 IN cause reduced accumulation of full-length cDNA in the viruslike particles. We show that the reduction in cDNA is accompanied by reduced amounts of early intermediates such as minus-strand, strong-stop DNA. Expression of a capsid (CA)-IN fusion protein (CA-IN) complemented catalytic site and nuclear localization mutants, but not DNA mutants. However, expression of a fusion of CA, reverse transcriptase (RT), and IN (CA-RT-IN) complemented transposition of catalytic site and nuclear localization signal mutants, increased the amount of cDNA in some of the mutants, and complemented transposition of several mutants to low frequencies. Expression of a CA-RT-IN protein with a Ty3 IN catalytic site mutation did not complement transposition of either a Ty3 catalytic site mutant or a nuclear localization mutant but did increase the amount of cDNA in several mutants and complement at least one of the cDNA mutants for transposition. These in vivo data support a model in which independent IN domains can contribute to reverse transcription and integration. We conclude that during reverse transcription, the Ty3 IN domain interacts closely with the polymerase domain and may even constitute a domain within a heterodimeric RT. These studies also suggest that during integration the IN catalytic site and at least portions of the IN carboxyl-terminal domain act in cis.