ABSTRACT
DNA variants that alter gene expression in trans are important sources of phenotypic variation. Nevertheless, the identity of trans-acting variants remains poorly understood. Single causal variants in several genes have been reported to affect the expression of numerous distant genes in trans. Whether these simple molecular architectures are representative of trans-acting variation is unknown. Here, we studied the large RAS signaling regulator gene IRA2, which contains variants with extensive trans-acting effects on gene expression in the yeast Saccharomyces cerevisiae. We used systematic CRISPR-based genome engineering and a sensitive phenotyping strategy to dissect causal variants to the nucleotide level. In contrast to the simple molecular architectures known so far, IRA2 contained at least seven causal nonsynonymous variants. The effects of these variants were modulated by nonadditive, epistatic interactions. Two variants at the 5'-end affected gene expression and growth only when combined with a third variant that also had no effect in isolation. Our findings indicate that the molecular basis of trans-acting genetic variation may be considerably more complex than previously appreciated.
Subject(s)
Saccharomyces cerevisiaeABSTRACT
Gene expression differences among individuals are shaped by trans-acting expression quantitative trait loci (eQTLs). Most trans-eQTLs map to hotspot locations that influence many genes. The molecular mechanisms perturbed by hotspots are often assumed to involve "vertical" cascades of effects in pathways that can ultimately affect the expression of thousands of genes. Here, we report that trans-eQTLs can affect the expression of adjacent genes via "horizontal" mechanisms that extend along a chromosome. Genes affected by trans-eQTL hotspots in the yeast Saccharomyces cerevisiae were more likely to be located next to each other than expected by chance. These paired hotspot effects tended to occur at adjacent genes that also show coexpression in response to genetic and environmental perturbations, suggesting shared mechanisms. Physical proximity and shared chromatin state, in addition to regulation of adjacent genes by similar transcription factors, were independently associated with paired hotspot effects among adjacent genes. Paired effects of trans-eQTLs can occur at neighboring genes even when these genes do not share a common function. This phenomenon could result in unexpected connections between regulatory genetic variation and phenotypes.
Subject(s)
Gene Expression Regulation, Fungal , Genetic Variation , Quantitative Trait Loci , Chromatin/genetics , Chromosomes/genetics , Saccharomyces cerevisiaeABSTRACT
Trans-acting DNA variants may specifically affect mRNA or protein levels of genes located throughout the genome. However, prior work compared trans-acting loci mapped in separate studies, many of which had limited statistical power. Here, we developed a CRISPR-based system for simultaneous quantification of mRNA and protein of a given gene via dual fluorescent reporters in single, live cells of the yeast Saccharomyces cerevisiae. In large populations of recombinant cells from a cross between two genetically divergent strains, we mapped 86 trans-acting loci affecting the expression of ten genes. Less than 20% of these loci had concordant effects on mRNA and protein of the same gene. Most loci influenced protein but not mRNA of a given gene. One locus harbored a premature stop variant in the YAK1 kinase gene that had specific effects on protein or mRNA of dozens of genes. These results demonstrate complex, post-transcriptional genetic effects on gene expression.
Subject(s)
Fungal Proteins/metabolism , RNA Processing, Post-Transcriptional/physiology , RNA, Messenger/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Single-Cell Analysis , DNA, Fungal/genetics , Fungal Proteins/genetics , Gene Expression Regulation, Fungal , Genetic Variation , Quantitative Trait Loci , RNA, Fungal/genetics , RNA, Fungal/metabolism , RNA, Messenger/geneticsABSTRACT
DNA variants that alter gene expression contribute to variation in many phenotypic traits. In particular, trans-acting variants, which are often located on different chromosomes from the genes they affect, are an important source of heritable gene expression variation. However, our knowledge about the identity and mechanism of causal trans-acting variants remains limited. Here, we developed a fine-mapping strategy called CRISPR-Swap and dissected three expression quantitative trait locus (eQTL) hotspots known to alter the expression of numerous genes in trans in the yeast Saccharomyces cerevisiae. Causal variants were identified by engineering recombinant alleles and quantifying the effects of these alleles on the expression of a green fluorescent protein-tagged gene affected by the given locus in trans. We validated the effect of each variant on the expression of multiple genes by RNA-sequencing. The three variants differed in their molecular mechanism, the type of genes they reside in, and their distribution in natural populations. While a missense leucine-to-serine variant at position 63 in the transcription factor Oaf1 (L63S) was almost exclusively present in the reference laboratory strain, the two other variants were frequent among S. cerevisiae isolates. A causal missense variant in the glucose receptor Rgt2 (V539I) occurred at a poorly conserved amino acid residue and its effect was strongly dependent on the concentration of glucose in the culture medium. A noncoding variant in the conserved fatty acid regulated (FAR) element of the OLE1 promoter influenced the expression of the fatty acid desaturase Ole1 in cis and, by modulating the level of this essential enzyme, other genes in trans. The OAF1 and OLE1 variants showed a non-additive genetic interaction, and affected cellular lipid metabolism. These results demonstrate that the molecular basis of trans-regulatory variation is diverse, highlighting the challenges in predicting which natural genetic variants affect gene expression.
Subject(s)
DNA-Binding Proteins/genetics , Evolution, Molecular , Regulatory Sequences, Nucleic Acid/genetics , Saccharomyces cerevisiae Proteins/genetics , Stearoyl-CoA Desaturase/genetics , Transcription Factors/genetics , CRISPR-Cas Systems/genetics , Fatty Acids/genetics , Fatty Acids/metabolism , Gene Expression Regulation, Fungal/genetics , Green Fluorescent Proteins/genetics , Lipid Metabolism/genetics , Monosaccharide Transport Proteins/genetics , Mutation, Missense/genetics , Phenotype , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolismABSTRACT
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.
Subject(s)
Retroelements/genetics , Ribosomal Proteins/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Conserved Sequence/genetics , Gene Expression Regulation, Fungal/drug effects , Phenotype , Protein Isoforms/genetics , RNA, Small Nucleolar/genetics , Tunicamycin/pharmacologyABSTRACT
Long-terminal repeat (LTR)-retrotransposons generate a copy of their DNA (cDNA) by reverse transcription of their RNA genome in cytoplasmic nucleocapsids. They are widespread in the eukaryotic kingdom and are the evolutionary progenitors of retroviruses. The Ty1 element of the budding yeast Saccharomyces cerevisiae was the first LTR-retrotransposon demonstrated to mobilize through an RNA intermediate, and not surprisingly, is the best studied. The depth of our knowledge of Ty1 biology stems not only from the predominance of active Ty1 elements in the S. cerevisiae genome but also the ease and breadth of genomic, biochemical, and cell biology approaches available to study cellular processes in yeast. This review describes the basic structure of Ty1 and its gene products, the replication cycle, the rapidly expanding compendium of host cofactors known to influence retrotransposition, and the nature of Ty1's elaborate symbiosis with its host. Our goal is to illuminate the value of Ty1 as a paradigm to explore the biology of LTR-retrotransposons in multicellular organisms, where the low frequency of retrotransposition events presents a formidable barrier to investigations of retrotransposon biology.
Subject(s)
DNA Replication , Retroelements , Saccharomyces cerevisiae/genetics , Genes, Fungal , Recombination, Genetic , Reverse TranscriptionABSTRACT
Long-terminal repeat (LTR)-retrotransposons generate a copy of their DNA (cDNA) by reverse transcription of their RNA genome in cytoplasmic nucleocapsids. They are widespread in the eukaryotic kingdom and are the evolutionary progenitors of retroviruses [1]. The Ty1 element of the budding yeast Saccharomyces cerevisiae was the first LTR-retrotransposon demonstrated to mobilize through an RNA intermediate, and not surprisingly, is the best studied. The depth of our knowledge of Ty1 biology stems not only from the predominance of active Ty1 elements in the S. cerevisiae genome but also the ease and breadth of genomic, biochemical and cell biology approaches available to study cellular processes in yeast. This review describes the basic structure of Ty1 and its gene products, the replication cycle, the rapidly expanding compendium of host co-factors known to influence retrotransposition and the nature of Ty1's elaborate symbiosis with its host. Our goal is to illuminate the value of Ty1 as a paradigm to explore the biology of LTR-retrotransposons in multicellular organisms, where the low frequency of retrotransposition events presents a formidable barrier to investigations of retrotransposon biology.
ABSTRACT
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.
Subject(s)
Cell Nucleus/genetics , Cell Nucleus/metabolism , Introns , RNA Precursors/genetics , RNA Precursors/metabolism , Base Pairing , Base Sequence , Cytoplasm/genetics , Cytoplasm/metabolism , Endoribonucleases/genetics , Endoribonucleases/metabolism , Evolution, Molecular , Exons , Gene Expression , Gene Silencing , Humans , Nucleic Acid Conformation , Poly(A)-Binding Proteins/genetics , Poly(A)-Binding Proteins/metabolism , RNA Precursors/chemistry , RNA Splicing , RNA, Fungal/genetics , RNA, Fungal/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Spliceosomes/genetics , Spliceosomes/metabolismABSTRACT
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.
Subject(s)
Endoplasmic Reticulum/genetics , Genome, Viral/genetics , Protein Biosynthesis/genetics , RNA/genetics , Retroelements/genetics , Cell Nucleus/genetics , Endoplasmic Reticulum/metabolism , RNA, Messenger/genetics , Ribosomes/genetics , Saccharomyces cerevisiae , Signal Recognition ParticleABSTRACT
Export of messenger RNA (mRNA) from the nucleus to the cytoplasm is a critical step in the gene expression pathway of eukaryotic cells. Here, we report the functional and structural characterization of the mammalian TREX-2 complex and show how it links transcription/processing with nuclear mRNA export. Mammalian TREX-2 is based on a germinal-centre associated nuclear protein (GANP) scaffold to which ENY2, PCID2 and centrins bind and depletion of any of these components inhibits mRNA export. The crystal structure of the GANP:ENY2 complex shows that two ENY2 chains interact directly with GANP, but they have different orientations from those observed on yeast Sac3. GANP is required to recruit ENY2 to nuclear pore complexes (NPCs), but ENY2 is not necessary to recruit GANP, which requires both its CID and MCM3AP domains, together with nucleoporin Nup153. GANP and ENY2 associate with RNA polymerase II and inhibition of mRNA processing redistributes GANP from NPCs into nuclear foci indicating that mammalian TREX-2 is associated with transcription. Thus, we implicate TREX-2 as an integral component of the mammalian mRNA export machinery where it links transcription and nuclear export by facilitating the transfer of mature mRNPs from the nuclear interior to NPCs.
Subject(s)
Acetyltransferases/chemistry , Cell Nucleus/genetics , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , RNA, Messenger/metabolism , Transcription, Genetic , Acetyltransferases/analysis , Acetyltransferases/metabolism , Active Transport, Cell Nucleus , Amino Acid Sequence , Cell Nucleus/metabolism , Crystallography, X-Ray , Exodeoxyribonucleases/metabolism , Humans , Intracellular Signaling Peptides and Proteins , Models, Molecular , Molecular Sequence Data , Nuclear Pore Complex Proteins/metabolism , Nuclear Proteins/analysis , Phosphoproteins/metabolism , RNA Transport , Transcription Factors/chemistry , Transcription Factors/metabolismABSTRACT
Sus1 is an evolutionary conserved protein that functions both in transcription and mRNA export and has been proposed to contribute to coupling these processes in yeast. Sus1 mediates its different roles as a component of both the histone H2B deubiquitinating module (Sus1-Sgf11-Ubp8-Sgf73) of the SAGA (Spt-Ada-Gcn5 acetyltransferase) transcriptional co-activator and the mRNA export complex, TREX-2 (Sus1-Sac3-Thp1-Cdc31). We have dissected the different functions of Sus1 with respect to its partitioning in transcription and export complexes using a mutational approach. Here we show that the sus1-10 (E18A, S19A, and G20A) and sus1-12 (V73A and D75A) alleles of Sus1 can be dissociated from TREX-2 while leaving its interaction with SAGA largely intact. Conversely, the binding to both TREX-2 and SAGA was impaired in the sus1-11 allele (G37A and W38A), in which two highly conserved residues were mutated. In vitro experiments demonstrated that dissociation of mutant Sus1 from its partners is caused by a reduced affinity toward the TREX-2 subunit, Sac3, and the SAGA factor, Sgf11, respectively. Consistent with the biochemical data, these sus1 mutant alleles showed differential genetic relationships with SAGA and mRNA export mutants. In vivo, all three sus1 mutants were impaired in targeting TREX-2 (i.e. Sac3) to the nuclear pore complexes and exhibited nuclear mRNA export defects. This study has implications for how Sus1, in combination with distinct interaction partners, can regulate diverse aspects of gene expression.
Subject(s)
Gene Expression Regulation, Fungal/physiology , Histones/metabolism , Nuclear Pore/metabolism , Nuclear Proteins/metabolism , RNA Transport/physiology , RNA, Fungal/metabolism , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Amino Acid Substitution , Histones/genetics , Multienzyme Complexes/genetics , Multienzyme Complexes/metabolism , Mutation, Missense , Nuclear Pore/genetics , Nuclear Proteins/genetics , RNA, Fungal/genetics , RNA, Messenger/genetics , RNA-Binding Proteins/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Trans-Activators/genetics , Trans-Activators/metabolism , Ubiquitination/physiologyABSTRACT
The yeast Sac3:Cdc31:Sus1:Thp1 (TREX-2) complex facilitates the repositioning and association of actively transcribing genes with nuclear pores (NPCs)-"gene gating"-that is central to integrating transcription, processing, and mRNA nuclear export. We present here the crystal structure of Sus1 and Cdc31 bound to a central region of Sac3 (the CID domain) that is crucial for its function. Sac3(CID) forms a long, gently undulating alpha helix around which one Cdc31 and two Sus1 chains are wrapped. Sus1 has an articulated helical hairpin fold that facilitates its wrapping around Sac3. In vivo studies using engineered mutations that selectively disrupted binding of individual chains to Sac3 indicated that Sus1 and Cdc31 function synergistically to promote NPC association of TREX-2 and mRNA nuclear export. These data indicate Sac3(CID) provides a scaffold within TREX-2 to integrate interactions between protein complexes to facilitate the coupling of transcription and mRNA export during gene expression.
Subject(s)
Calcium-Binding Proteins/chemistry , Cell Cycle Proteins/chemistry , Nuclear Pore/metabolism , Nuclear Proteins/chemistry , RNA Transport , RNA, Messenger/metabolism , RNA-Binding Proteins/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/physiology , Amino Acid Sequence , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Crystallography, X-Ray , In Situ Hybridization , Microscopy, Fluorescence , Molecular Sequence Data , Nuclear Pore/genetics , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Nucleocytoplasmic Transport Proteins , Porins , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Sequence Homology, Amino AcidABSTRACT
LINE-1 (L1) retrotransposons comprise approximately 17% of human DNA, yet little is known about L1 integration. Here, we characterized 100 retrotransposition events in HeLa cells and show that distinct DNA repair pathways can resolve L1 cDNA retrotransposition intermediates. L1 cDNA resolution can lead to various forms of genetic instability including the generation of chimeric L1s, intrachromosomal deletions, intrachromosomal duplications, and intra-L1 rearrangements as well as a possible interchromosomal translocation. The L1 retrotransposition machinery also can mobilize U6 snRNA to new genomic locations, increasing the repertoire of noncoding RNAs that are mobilized by L1s. Finally, we have determined that the L1 reverse transcriptase can faithfully replicate its own transcript and has a base misincorporation error rate of approximately 1/7,000 bases. These data indicate that L1 retrotransposition in transformed human cells can lead to a variety of genomic rearrangements and suggest that host processes act to restrict L1 integration in cultured human cells. Indeed, the initial steps in L1 retrotransposition may define a host/parasite battleground that serves to limit the number of active L1s in the genome.
Subject(s)
Long Interspersed Nucleotide Elements/genetics , Cells, Cultured , Consensus Sequence/genetics , DNA, Complementary/genetics , Genetic Engineering , Genome, Human , Humans , Models, Genetic , Mutagenesis, Insertional/genetics , Mutation/genetics , Nucleotides/genetics , Substrate Specificity , Translocation, Genetic/geneticsABSTRACT
De novo LINE-1 (long interspersed element-1, or L1) retrotransposition events are responsible for approximately 1/1,000 disease-causing mutations in humans. Previously, L1.2 was identified as the likely progenitor of a mutagenic insertion in the factor VIII gene in a patient with hemophilia A. It subsequently was shown to be one of a small number of active L1s in the human genome. Here, we demonstrate that L1.2 is present at an intermediate insertion allele frequency in worldwide human populations and that common alleles (L1.2A and L1.2B) exhibit an approximately 16-fold difference in their ability to retrotranspose in cultured human HeLa cells. Chimera analysis revealed that two amino acid substitutions (S1259L and I1220M) downstream of the conserved cysteine-rich motif in L1 open reading frame 2 are largely responsible for the observed reduction in L1.2A retrotransposition efficiency. Thus, common L1 alleles can vary widely in their retrotransposition potential. We propose that such allelic heterogeneity can influence the potential L1 mutational load present in an individual genome.