The Mediator co-activator complex regulates Ty1 retromobility by controlling the balance between Ty1i and Ty1 promoters.

The Ty1 retrotransposons present in the genome of Saccharomyces cerevisiae belong to the large class of mobile genetic elements that replicate via an RNA intermediary and constitute a significant portion of most eukaryotic genomes. The retromobility of Ty1 is regulated by numerous host factors, including several subunits of the Mediator transcriptional co-activator complex. In spite of its known function in the nucleus, previous studies have implicated Mediator in the regulation of post-translational steps in Ty1 retromobility. To resolve this paradox, we systematically examined the effects of deleting non-essential Mediator subunits on the frequency of Ty1 retromobility and levels of retromobility intermediates. Our findings reveal that loss of distinct Mediator subunits alters Ty1 retromobility positively or negatively over a >10,000-fold range by regulating the ratio of an internal transcript, Ty1i, to the genomic Ty1 transcript. Ty1i RNA encodes a dominant negative inhibitor of Ty1 retromobility that blocks virus-like particle maturation and cDNA synthesis. These results resolve the conundrum of Mediator exerting sweeping control of Ty1 retromobility with only minor effects on the levels of Ty1 genomic RNA and the capsid protein, Gag. Since the majority of characterized intrinsic and extrinsic regulators of Ty1 retromobility do not appear to effect genomic Ty1 RNA levels, Mediator could play a central role in integrating signals that influence Ty1i expression to modulate retromobility.

Border collies of the genome: domestication of an autonomous retrovirus-like transposon.

Retrotransposons often spread rapidly through eukaryotic genomes until they are neutralized by host-mediated silencing mechanisms, reduced by recombination and mutation, and lost or transformed into benevolent entities. But the Ty1 retrotransposon appears to have been domesticated to guard the genome of Saccharomyces cerevisiae.

Global Epitranscriptomics Profiling of RNA Post-Transcriptional Modifications as an Effective Tool for Investigating the Epitranscriptomics of Stress Response.

The simultaneous detection of all the post-transcriptional modifications (PTMs) that decorate cellular RNA can provide comprehensive information on the effects of changing environmental conditions on the entire epitranscriptome. To capture this type of information, we performed the analysis of ribonucleotide mixtures produced by hydrolysis of total RNA extracts from S. cerevisiae that was grown under hyperosmotic and heat shock conditions. Their global PTM profiles clearly indicated that the cellular responses to these types of stresses involved profound changes in the production of specific PTMs. The observed changes involved not only up-/down-regulation of typical PTMs, but also the outright induction of new ones that were absent under normal conditions, or the elimination of others that were normally present. Pointing toward the broad involvement of different classes of RNAs, many of the newly observed PTMs differed from those engaged in the known tRNA-based mechanism of translational recoding, which is induced by oxidative stress. Some of the expression effects were stress-specific, whereas others were not, thus suggesting that RNA PTMs may perform multifaceted activities in stress response, which are subjected to distinctive regulatory pathways. To explore their signaling networks, we implemented a strategy based on the systematic deletion of genes that connect established response genes with PTM biogenetic enzymes in a putative interactomic map. The results clearly identified PTMs that were under direct HOG control, a well-known protein kinase pathway involved in stress response in eukaryotes. Activation of this signaling pathway has been shown to result in the stabilization of numerous mRNAs and the induction of selected lncRNAs involved in chromatin remodeling. The fact that PTMs are capable of altering the activity of the parent RNAs suggest their possible participation in feedback mechanisms aimed at modulating the regulatory functions of such RNAs. This tantalizing hypothesis will be the object of future studies.

Nuclear expression of a group II intron is consistent with spliceosomal intron ancestry.

Group II introns are self-splicing RNAs found in eubacteria, archaea, and eukaryotic organelles. They are mechanistically similar to the metazoan nuclear spliceosomal introns; therefore, group II introns have been invoked as the progenitors of the eukaryotic pre-mRNA introns. However, the ability of group II introns to function outside of the bacteria-derived organelles is debatable, since they are not found in the nuclear genomes of eukaryotes. Here, we show that the Lactococcus lactis Ll.LtrB group II intron splices accurately and efficiently from different pre-mRNAs in a eukaryote, Saccharomyces cerevisiae. However, a pre-mRNA harboring a group II intron is spliced predominantly in the cytoplasm and is subject to nonsense-mediated mRNA decay (NMD), and the mature mRNA from which the group II intron is spliced is poorly translated. In contrast, a pre-mRNA bearing the Tetrahymena group I intron or the yeast spliceosomal ACT1 intron at the same location is not subject to NMD, and the mature mRNA is translated efficiently. Thus, a group II intron can splice from a nuclear transcript, but RNA instability and translation defects would have favored intron loss or evolution into protein-dependent spliceosomal introns, consistent with the bacterial group II intron ancestry hypothesis.

Co-translational localization of an LTR-retrotransposon RNA to the endoplasmic reticulum nucleates virus-like particle assembly sites.

The transcript of retrovirus-like transposons functions as an mRNA for synthesis of capsid and replication proteins and as the genomic RNA of virus-like particles (VLPs), wherein the genome is replicated. Retrotransposon RNA and proteins coalesce in a cytoplasmic focus, or retrosome, to initiate VLP assembly, but it is not known how the retrosome is nucleated. We determined how the RNA and Gag protein of the Saccharomyces cerevisiae Ty1 retrotransposon are directed to the retrosome. We found that Ty1 RNA is translated in association with signal recognition particle (SRP), a universally conserved chaperone that binds specific ribosome-nascent chain (RNC) complexes and targets the nascent peptide to the endoplasmic reticulum (ER). Gag is translocated to the ER lumen; yet, it is also found in the cytoplasm, associated with SRP-RNC complexes. In the absence of ER translocation, Gag is synthesized but rapidly degraded, and Ty1 RNA does not coalesce in retrosomes. These findings suggest that Gag adopts a stable conformation in the ER lumen, is retrotranslocated to the cytoplasm, binds to Ty1 RNA on SRP-RNC complexes and multimerizes to nucleate retrosomes. Consistent with this model, we show that slowing the rate of co-translational ER translocation by limiting SRP increases the prevalence of retrosomes, while suppressing the translocation defect of srp hypomorphs by slowing translational elongation rapidly decreases retrosome formation. Thus, retrosomes are dynamic foci of Ty1 RNA-RNC complexes whose formation is modulated by the rate of co-translational ER translocation. Together, these findings suggest that translating Ty1 mRNA and the genomic RNA of VLPs originate in a single pool and moreover, that co-translational localization of Ty1 RNA nucleates the presumptive VLP assembly site. The separation of nascent Gag from its RNA template by transit through the ER allows Gag to bind translating Ty1 RNA without displaying a cis-preference for its encoding RNA.

RNA-RNA interactions and pre-mRNA mislocalization as drivers of group II intron loss from nuclear genomes.

Group II introns are commonly believed to be the progenitors of spliceosomal introns, but they are notably absent from nuclear genomes. Barriers to group II intron function in nuclear genomes therefore beg examination. A previous study showed that nuclear expression of a group II intron in yeast results in nonsense-mediated decay and translational repression of mRNA, and that these roadblocks to expression are group II intron-specific. To determine the molecular basis for repression of gene expression, we investigated cellular dynamics of processed group II intron RNAs, from transcription to cellular localization. Our data show pre-mRNA mislocalization to the cytoplasm, where the RNAs are targeted to foci. Furthermore, tenacious mRNA-pre-mRNA interactions, based on intron-exon binding sequences, result in reduced abundance of spliced mRNAs. Nuclear retention of pre-mRNA prevents this interaction and relieves these expression blocks. In addition to providing a mechanistic rationale for group II intron-specific repression, our data support the hypothesis that RNA silencing of the host gene contributed to expulsion of group II introns from nuclear genomes and drove the evolution of spliceosomal introns.

A nucleosomal surface defines an integration hotspot for the Saccharomyces cerevisiae Ty1 retrotransposon.

Ty1, the most abundant retrotransposon in Saccharomyces cerevisiae, integrates preferentially upstream of genes transcribed by RNA polymerase III (Pol III). Targeting is likely due to interactions between the Ty1 integration complex and a feature of chromatin characteristic of sites of Pol III transcription. To better understand Ty1 targeting determinants, >150,000 Ty1 insertions were mapped onto the S. cerevisiae genome sequence. Logistic regression was used to assess relationships between patterns of Ty1 integration and various genomic features, including genome-wide data sets of histone modifications and transcription-factor binding sites. Nucleosomes were positively associated with Ty1 insertions, and fine-scale mapping of insertions upstream of genes transcribed by Pol III indicated that Ty1 preferentially integrates into nucleosome-bound DNA near the H2A/H2B interface. Outside of genes transcribed by Pol III, Ty1 avoids coding sequences, a pattern that is not due to selection, but rather reflects a preference for nucleosome-rich sites flanking genes. Ty1 insertion sites were also mapped in four mutant lines that affect Ty1 transposition frequency or integration specificity (rrm3Δ, hos2Δ, rtt109Δ, and rad6Δ). Patterns of integration were largely preserved in the mutants, although significantly more insertions into coding sequences were observed in the rad6Δ strain, suggesting a loosening of target specificity in this mutant that lacks an enzyme involved in ubiquitinating H2A. Overall, our data suggest that nucleosomes are necessary for Ty1 integration, and that a secondary factor, likely a histone modification or nucleosome-bound factor enriched at sites of Pol III transcription, determines preferred target sites.

Retrotransposition is associated with genome instability during chronological aging.

Genetic damage through mutations and genome rearrangements has been hypothesized to contribute to aging. The specific mechanisms responsible for age-induced increases in mutation and chromosome rearrangement frequencies and a potential causative role for DNA damage in aging are under active investigation. Retrotransposons are mobile genetic elements that cause insertion mutations and contribute to genome rearrangements through nonallelic recombination events in humans and other organisms. We have investigated the role of endogenous Ty1 retrotransposons in aging-associated increases in genome instability using the Saccharomyces cerevisiae chronological aging model. We show that age-induced increases in loss of heterozygosity and chromosome loss events are consistently diminished by mutations or treatments that reduce Ty1 retrotransposition. Ty1 mobility is elevated in very old yeast populations, and new retromobility events are often associated with chromosome rearrangements. These results reveal a correlation between retrotransposition and genome instability during yeast aging. Retrotransposition may contribute to genetic damage during aging in diverse organisms and provides a useful tool for studying whether genetic damage is a causative factor for aging.

The intra-S phase checkpoint protein Tof1 collaborates with the helicase Rrm3 and the F-box protein Dia2 to maintain genome stability in Saccharomyces cerevisiae.

The intra-S phase checkpoint protein complex Tof1/Csm3 of Saccharomyces cerevisiae antagonizes Rrm3 helicase to modulate replication fork arrest not only at the replication termini of rDNA but also at strong nonhistone protein binding sites throughout the genome. We investigated whether these checkpoint proteins acted either antagonistically or synergistically with Rrm3 in mediating other important functions such as maintenance of genome stability. High retromobility of a normally quiescent retrovirus-like transposable element Ty1 of S. cerevisiae is a form of genome instability, because the transposition events induce mutations. We measured the transposition of Ty1 in various genetic backgrounds and discovered that Tof1 suppressed excessive retromobility in collaboration with either Rrm3 or the F-box protein Dia2. Although both Rrm3 and Dia2 are believed to facilitate fork movement, fork stalling at DNA-protein complexes did not appear to be a major contributor to enhancement of retromobility. Absence of the aforementioned proteins either individually or in pair-wise combinations caused karyotype changes as revealed by the altered migrations of the individual chromosomes in pulsed field gels. The mobility changes were RNase H-resistant and therefore, unlikely to have been caused by extensive R loop formation. These mutations also resulted in alterations of telomere lengths. However, the latter changes could not fully account for the magnitude of the observed karyotypic alterations. We conclude that unlike other checkpoint proteins that are known to be required for elevated retromobility, Tof1 suppressed high frequency retrotransposition and maintained karyotype stability in collaboration with the aforementioned proteins.

Reliance of Host-Encoded Regulators of Retromobility on Ty1 Promoter Activity or Architecture.

The Ty1 retrotransposon family is maintained in a functional but dormant state by its host, Saccharomyces cerevisiae. Several hundred RHF and RTT genes encoding co-factors and restrictors of Ty1 retromobility, respectively, have been identified. Well-characterized examples include MED3 and MED15, encoding subunits of the Mediator transcriptional co-activator complex; control of retromobility by Med3 and Med15 requires the Ty1 promoter in the U3 region of the long terminal repeat. To characterize the U3-dependence of other Ty1 regulators, we screened a library of 188 known rhf and rtt mutants for altered retromobility of Ty1his3AI expressed from the strong, TATA-less TEF1 promoter or the weak, TATA-containing U3 promoter. Two classes of genes, each including both RHFs and RTTs, were identified. The first class comprising 82 genes that regulated Ty1his3AI retromobility independently of U3 is enriched for RHF genes that restrict the G1 phase of the cell cycle and those involved in transcriptional elongation and mRNA catabolism. The second class of 51 genes regulated retromobility of Ty1his3AI driven only from the U3 promoter. Nineteen U3-dependent regulators (U3DRs) also controlled retromobility of Ty1his3AI driven by the weak, TATA-less PSP2 promoter, suggesting reliance on the low activity of U3. Thirty-one U3DRs failed to modulate P PSP2 -Ty1his3AI retromobility, suggesting dependence on the architecture of U3. To further investigate the U3-dependency of Ty1 regulators, we developed a novel fluorescence-based assay to monitor expression of p22-Gag, a restriction factor expressed from the internal Ty1i promoter. Many U3DRs had minimal effects on levels of Ty1 RNA, Ty1i RNA or p22-Gag. These findings uncover a role for the Ty1 promoter in integrating signals from diverse host factors to modulate Ty1 RNA biogenesis or fate.

5' to 3' mRNA decay factors colocalize with Ty1 gag and human APOBEC3G and promote Ty1 retrotransposition.

The genomic RNA of retroviruses and retrovirus-like transposons must be sequestered from the cellular translational machinery so that it can be packaged into viral particles. Eukaryotic mRNA processing bodies (P bodies) play a central role in segregating cellular mRNAs from the translational machinery for storage or decay. In this work, we provide evidence that the RNA of the Saccharomyces cerevisiae Ty1 retrotransposon is packaged into virus-like particles (VLPs) in P bodies. Ty1 RNA is translationally repressed, and Ty1 Gag, the capsid and RNA binding protein, accumulates in discrete cytoplasmic foci, a subset of which localize to P bodies. Human APOBEC3G, a potent Ty1 restriction factor that is packaged into Ty1 VLPs via an interaction with Gag, also localizes to P bodies. The association of APOBEC3G with P bodies does not require Ty1 element expression, suggesting that P-body localization of APOBEC3G and Ty1 Gag precedes VLP assembly. Additionally, we report that two P-body-associated 5' to 3' mRNA decay pathways, deadenylation-dependent mRNA decay (DDD) and nonsense-mediated decay (NMD), stimulate Ty1 retrotransposition. The additive contributions of DDD and NMD explain the strong requirement for general 5' to 3' mRNA degradation factors Dcp1, Dcp2, and Xrn1 in Ty1 retromobility. 5' to 3' decay factors act at a posttranslational step in retrotransposition, and Ty1 RNA packaging into VLPs is abolished in the absence of the 5' to 3' exonuclease Xrn1. Together, the results suggest that VLPs assemble in P bodies and that 5' to 3' mRNA decay is essential for the packaging of Ty1 RNA in VLPs.

Rrm3 protects the Saccharomyces cerevisiae genome from instability at nascent sites of retrotransposition.

The DNA helicase Rrm3 promotes replication fork progression through >1000 discrete genomic regions and represses the cDNA-mediated mobility of the Ty1 retrotransposon. We explored the connection between DNA replication and Ty1 retromobility by investigating the basis of increased retromobility in an rrm3 mutant. Even though Ty1 cDNA levels are increased in the absence of RRM3, neither the level nor target-site specificity of cDNA integration was altered. Instead, cDNA was incorporated into the genome by a Rad52-dependent mechanism that did not involve gene conversion of genomic Ty1 sequences. In rrm3 isolates, incorporated cDNA was often present in tandem arrays. Multimeric cDNA arrays probably arise during chromosomal break repair, since their appearance was strongly correlated with the formation of gross chromosomal rearrangements. Moreover, Ty1 multimers were invariantly located on rearranged chromosomes, when present. Overexpression of a cellular RNase H, which degrades RNA in an RNA:DNA hybrid, completely suppressed the increase in Ty1 multimer formation in an rrm3 mutant. We propose that RNA:DNA hybrid regions within nascent retrotransposition events block replication in an rrm3 mutant, leading to chromosome breaks within Ty1 sequences. Multiple extragenomic Ty1 cDNA molecules are then used as donors in recombinational repair of the break before it is healed.

Incorporation of Y'-Ty1 cDNA destabilizes telomeres in Saccharomyces cerevisiae telomerase-negative mutants.

Ty1 retrotransposons in Saccharomyces cerevisiae are activated by telomere erosion. Ty1-dependent reverse transcription of mRNA from subtelomeric Y' repeats generates chimeric Y'-Ty1 cDNA. Here, we show that Y'-Ty1 cDNA is incorporated at eroding telomeres in the absence of telomerase. Telomeric incorporation of Y'-Ty1 cDNA promotes genome rearrangements.

Inhibition of a yeast LTR retrotransposon by human APOBEC3 cytidine deaminases.

The mammalian APOBEC3 family of cytidine deaminases includes several members that possess potent antiretroviral activity. Human APOBEC3F and APOBEC3G are specifically incorporated into human immunodeficiency virus type 1 (HIV-1) progeny virions in the absence of virion infectivity factor (Vif), where they deaminate deoxycytidine to deoxyuridine on the minus strand of nascent reverse transcripts. Editing of the HIV-1 cDNA leads to its degradation or to G to A hypermutation of the integrated provirus. Here, we show that APOBEC3 proteins also restrict the activity of a distantly related long terminal repeat (LTR) retrotransposon. When expressed in the yeast Saccharomyces cerevisiae, human APOBEC3C, APOBEC3F, or APOBEC3G or mouse APOBEC3 potently inhibit replication of the Ty1 LTR retrotransposon. APOBEC3G interacts with Ty1 Gag and is packaged into Ty1 virus-like particles (VLPs) by a mechanism that closely resembles the one it uses to enter HIV-1 virions. Expression of APOBEC3G results in a reduced level of Ty1 cDNA integration and G to A editing of integrated Ty1 cDNA. Our findings indicate that APOBEC3G restricts Ty1 and HIV-1 by similar mechanisms and suggest that the APOBEC3 proteins target a substantially broader spectrum of retroelements than previously appreciated.

Evidence that Set1, a factor required for methylation of histone H3, regulates rDNA silencing in S. cerevisiae by a Sir2-independent mechanism.

Several types of histone modifications have been shown to control transcription. Recent evidence suggests that specific combinations of these modifications determine particular transcription patterns. The histone modifications most recently shown to play critical roles in transcription are arginine-specific and lysine-specific methylation. Lysine-specific histone methyltransferases all contain a SET domain, a conserved 130 amino acid motif originally identified in polycomb- and trithorax-group proteins from Drosophila. Members of the SU(VAR)3-9 family of SET-domain proteins methylate K9 of histone H3. Methylation of H3 has also been shown to occur at K4. Several studies have suggested a correlation between K4-methylated H3 and active transcription. In this paper, we provide evidence that K4-methylated H3 is required in a negative role, rDNA silencing in Saccharomyces cerevisiae. In a screen for rDNA silencing mutants, we identified a mutation in SET1, previously shown to regulate silencing at telomeres and HML. Recent work has shown that Set1 is a member of a complex and is required for methylation of K4 of H3 at several genomic locations. In addition, we demonstrate that a K4R change in H3, which prevents K4 methylation, impairs rDNA silencing, indicating that Set1 regulates rDNA silencing, directly or indirectly, via H3 methylation. Furthermore, we present several lines of evidence that the role of Set1 in rDNA silencing is distinct from that of the histone deacetylase Sir2. Together, these results suggest that Set1-dependent H3 methylation is required for rDNA silencing in a Sir2-independent fashion.

Hos2 and Set3 promote integration of Ty1 retrotransposons at tRNA genes in Saccharomyces cerevisiae.

The yeast LTR retrotransposon Ty1 integrates preferentially into regions upstream of tRNA genes. The chromatin structure of transcriptionally active tRNA genes is known to be important for Ty1 integration, but specific chromatin factors that enhance integration at tRNA genes have not been identified. Here we report that the histone deacetylase, Hos2, and the Trithorax-group protein, Set3, both components of the Set3 complex (Set3C), enhance transposition of chromosomal Ty1 elements by promoting integration into the upstream region of tRNA genes. Deletion of HOS2 or SET3 reduced the mobility of a chromosomal Ty1his3AI element about sevenfold. Despite the fact that Ty1his3AI RNA, total Ty1 RNA, and total Ty1 cDNA levels were not reduced in hos2delta or set3delta mutants, transposition of endogenous Ty1 elements into the upstream regions of tRNA(Gly) genes was substantially decreased. Furthermore, when equivalent numbers of Ty1HIS3 mobility events launched from a pGAL1:Ty1his3AI plasmid were analyzed, only one-quarter to one-half as many were found upstream of tRNA(Gly) genes in a hos2delta or set3delta mutant than in a wild-type strain. Chromatin immunoprecipitation analysis revealed that Hos2 is physically associated with tRNA genes. Taken together, our results support the hypothesis that Hos2 and Set3 function at tRNA genes to promote Ty1 integration.

Ty1 mobilizes subtelomeric Y' elements in telomerase-negative Saccharomyces cerevisiae survivors.

When telomerase is inactivated in Saccharomyces cerevisiae, telomeric DNA shortens with every cell division, and cells stop dividing after approximately 100 generations. Survivors that form in these senescent populations and resume growing have variably amplified arrays of subtelomeric Y' elements. We marked a chromosomal Y' element with the his3AI retrotransposition indicator gene and found that Y'HIS3 cDNA was incorporated into the genome at approximately 10- to 1,000-fold-higher frequencies in survivors compared to telomerase-positive strains. Y'HIS3 cDNA mobility was significantly reduced if assayed at 30 degrees C, a nonpermissive temperature for Ty1 retrotransposition, or in the absence of Tec1p, a transcription factor for Ty1. Microarray analysis revealed that Y' RNA is preferentially associated with Ty1 virus-like particles (VLPs). Genomic copies of Y'HIS3 cDNA typically have downstream oligo(A) tracts, followed by a complete Ty1 long terminal repeat and TYA1 or TYB1 sequences. These data are consistent with the use of Ty1 cDNA to prime reverse transcription of polyadenylated Y' RNA within Ty1 VLPs. Unmarked Y'-oligo(A)-Ty1 cDNA was also detected in survivors, reaching copy numbers of approximately 10(-2) per genome. We propose that Y'-oligo(A)-Ty1 cDNA recombines with Y' elements at eroding telomeres in survivors and may play a role in telomere maintenance in the absence of telomerase.

Paralog-Specific Functions of RPL7A and RPL7B Mediated by Ribosomal Protein or snoRNA Dosage in Saccharomyces cerevisiae.

Most ribosomal proteins in Saccharomyces cerevisiae are encoded by two paralogs that additively produce the optimal protein level for cell growth. Nonetheless, deleting one paralog of most ribosomal protein gene pairs results in a variety of phenotypes not observed when the other paralog is deleted. To determine whether paralog-specific phenotypes associated with deleting RPL7A or RPL7B stem from distinct functions or different levels of the encoded isoforms, the coding region and introns of one paralog, including an intron-embedded snoRNA (small nucleolar RNA) gene, were exchanged with that of the other paralog. Among mutants harboring a single native or chimeric RPL7 allele, expression from the RPL7A locus exceeded that from the RPL7B locus, and more Rpl7a was expressed from either locus than Rpl7b Phenotypic differences in tunicamycin sensitivity, ASH1 mRNA localization, and mobility of the Ty1 retrotransposon were strongly correlated with Rpl7 and ribosome levels, but not with the Rpl7 or snoRNA isoform expressed. Although Ty1 RNA is cotranslationally localized, depletion of Rpl7 minimally affected synthesis of Ty1 Gag protein, but strongly influenced Ty1 RNA localization. Unlike the other processes studied, Ty1 cDNA accumulation was influenced by both the level and isoform of Rpl7 or snoRNA expressed. These cellular processes had different minimal threshold values for Rpl7 and ribosome levels, but all were functional when isoforms of either paralog were expressed from the RPL7A locus or both RPL7 loci. This study illustrates the broad range of phenotypes that can result from depleting ribosomes to different levels.

Structure-Function Model for Kissing Loop Interactions That Initiate Dimerization of Ty1 RNA.

The genomic RNA of the retrotransposon Ty1 is packaged as a dimer into virus-like particles. The 5' terminus of Ty1 RNA harbors cis-acting sequences required for translation initiation, packaging and initiation of reverse transcription (TIPIRT). To identify RNA motifs involved in dimerization and packaging, a structural model of the TIPIRT domain in vitro was developed from single-nucleotide resolution RNA structural data. In general agreement with previous models, the first 326 nucleotides of Ty1 RNA form a pseudoknot with a 7-bp stem (S1), a 1-nucleotide interhelical loop and an 8-bp stem (S2) that delineate two long, structured loops. Nucleotide substitutions that disrupt either pseudoknot stem greatly reduced helper-Ty1-mediated retrotransposition of a mini-Ty1, but only mutations in S2 destabilized mini-Ty1 RNA in cis and helper-Ty1 RNA in trans. Nested in different loops of the pseudoknot are two hairpins with complementary 7-nucleotide motifs at their apices. Nucleotide substitutions in either motif also reduced retrotransposition and destabilized mini- and helper-Ty1 RNA. Compensatory mutations that restore base-pairing in the S2 stem or between the hairpins rescued retrotransposition and RNA stability in cis and trans. These data inform a model whereby a Ty1 RNA kissing complex with two intermolecular kissing-loop interactions initiates dimerization and packaging.