Meiotic cDNA libraries reveal gene truncations and mitochondrial proteins important for competitive fitness in Saccharomyces cerevisiae.

Gametogenesis is an evolutionarily conserved developmental program whereby a diploid progenitor cell undergoes meiosis and cellular remodeling to differentiate into haploid gametes, the precursors for sexual reproduction. Even in the simple eukaryotic organism Saccharomyces cerevisiae, the meiotic transcriptome is very rich and complex, thereby necessitating new tools for functional studies. Here, we report the construction of 5 stage-specific, inducible complementary DNA libraries from meiotic cells that represent over 84% of the genes found in the budding yeast genome. We employed computational strategies to detect endogenous meiotic transcript isoforms as well as library-specific gene truncations. Furthermore, we developed a robust screening pipeline to test the effect of each complementary DNA on competitive fitness. Our multiday proof-of-principle time course revealed 877 complementary DNAs that were detrimental for competitive fitness when overexpressed. The list included mitochondrial proteins that cause dose-dependent disruption of cellular respiration as well as library-specific gene truncations that expose a dominant negative effect on competitive growth. Together, these high-quality complementary DNA libraries provide an important tool for systematically identifying meiotic genes, transcript isoforms, and protein domains that are important for a specific biological function.

Global mapping of translation initiation sites by TIS profiling in budding yeast.

Translation initiation site (TIS) profiling allows for the genome-wide identification of TISs in vivo by exclusively capturing mRNA fragments within ribosomes that have just completed translation initiation. It leverages translation inhibitors, such as harringtonine and lactimidomycin (LTM), that preferentially capture ribosomes at start codon positions, protecting TIS-derived mRNA fragments from nuclease digestion. Here, we describe a step-by-step protocol for TIS profiling in LTM-treated budding yeast that we developed to identify TISs and open reading frames in vegetative and meiotic cells. For complete details on the use and execution of this protocol, please refer to Eisenberg et al. (2020).

Translation Initiation Site Profiling Reveals Widespread Synthesis of Non-AUG-Initiated Protein Isoforms in Yeast.

Genomic analyses in budding yeast have helped define the foundational principles of eukaryotic gene expression. However, in the absence of empirical methods for defining coding regions, these analyses have historically excluded specific classes of possible coding regions, such as those initiating at non-AUG start codons. Here, we applied an experimental approach to globally annotate translation initiation sites in yeast and identified 149 genes with alternative N-terminally extended protein isoforms initiating from near-cognate codons upstream of annotated AUG start codons. These isoforms are produced in concert with canonical isoforms and translated with high specificity, resulting from initiation at only a small subset of possible start codons. The non-AUG initiation driving their production is enriched during meiosis and induced by low eIF5A, which is seen in this context. These findings reveal widespread production of non-canonical protein isoforms and unexpected complexity to the rules by which even a simple eukaryotic genome is decoded.

Evidence for an Integrated Gene Repression Mechanism Based on mRNA Isoform Toggling in Human Cells.

We recently described an unconventional mode of gene regulation in budding yeast by which transcriptional and translational interference collaborate to down-regulate protein expression. Developmentally timed transcriptional interference inhibited production of a well translated mRNA isoform and resulted in the production of an mRNA isoform containing inhibitory upstream open reading frames (uORFs) that prevented translation of the main ORF. Transcriptional interference and uORF-based translational repression are established mechanisms outside of yeast, but whether this type of integrated regulation was conserved was unknown. Here we find that, indeed, a similar type of regulation occurs at the locus for the human oncogene MDM2 We observe evidence of transcriptional interference between the two MDM2 promoters, which produce a poorly translated distal promoter-derived uORF-containing mRNA isoform and a well-translated proximal promoter-derived transcript. Down-regulation of distal promoter activity markedly up-regulates proximal promoter-driven expression and results in local reduction of histone H3K36 trimethylation. Moreover, we observe that this transcript toggling between the two MDM2 isoforms naturally occurs during human embryonic stem cell differentiation programs.

Strategies and Challenges in Identifying Function for Thousands of sORF-Encoded Peptides in Meiosis.

Recent genomic analyses have revealed pervasive translation from formerly unrecognized short open reading frames (sORFs) during yeast meiosis. Despite their short length, which has caused these regions to be systematically overlooked by traditional gene annotation approaches, meiotic sORFs share many features with classical genes, implying the potential for similar types of cellular functions. We found that sORF expression accounts for approximately 10-20% of the cellular translation capacity in yeast during meiotic differentiation and occurs within well-defined time windows, suggesting the production of relatively abundant peptides with stage-specific meiotic roles from these regions. Here, we provide arguments supporting this hypothesis and discuss sORF similarities and differences, as a group, to traditional protein coding regions, as well as challenges in defining their specific functions.

Pathophysiological consequences and benefits of HFE mutations: 20 years of research.

Mutations in the HFE (hemochromatosis) gene cause hereditary hemochromatosis, an iron overload disorder that is hallmarked by excessive accumulation of iron in parenchymal organs. The HFE mutation p.Cys282Tyr is pathologically most relevant and occurs in the Caucasian population with a carrier frequency of up to 1 in 8 in specific European regions. Despite this high prevalence, the mutation causes a clinically relevant phenotype only in a minority of cases. In this review, we summarize historical facts and recent research findings about hereditary hemochromatosis, and outline the pathological consequences of the associated gene defects. In addition, we discuss potential advantages of HFE mutations in asymptomatic carriers.

Exon Junction Complexes Show a Distributional Bias toward Alternatively Spliced mRNAs and against mRNAs Coding for Ribosomal Proteins.

The exon junction complex (EJC) connects spliced mRNAs to posttranscriptional processes including RNA localization, transport, and regulated degradation. Here, we provide a comprehensive analysis of bona fide EJC binding sites across the transcriptome including all four RNA binding EJC components eIF4A3, BTZ, UPF3B, and RNPS1. Integration of these data sets permits definition of high-confidence EJC deposition sites as well as assessment of whether EJC heterogeneity drives alternative nonsense-mediated mRNA decay pathways. Notably, BTZ (MLN51 or CASC3) emerges as the EJC subunit that is almost exclusively bound to sites 20-24 nucleotides upstream of exon-exon junctions, hence defining EJC positions. By contrast, eIF4A3, UPF3B, and RNPS1 display additional RNA binding sites suggesting accompanying non-EJC functions. Finally, our data show that EJCs are largely distributed across spliced RNAs in an orthodox fashion, with two notable exceptions: an EJC deposition bias in favor of alternatively spliced transcripts and against the mRNAs that encode ribosomal proteins.

The differential expression of alternatively polyadenylated transcripts is a common stress-induced response mechanism that modulates mammalian mRNA expression in a quantitative and qualitative fashion.

Stress adaptation plays a pivotal role in biological processes and requires tight regulation of gene expression. In this study, we explored the effect of cellular stress on mRNA polyadenylation and investigated the implications of regulated polyadenylation site usage on mammalian gene expression. High-confidence polyadenylation site mapping combined with global pre-mRNA and mRNA expression profiling revealed that stress induces an accumulation of genes with differentially expressed polyadenylated mRNA isoforms in human cells. Specifically, stress provokes a global trend in polyadenylation site usage toward decreased utilization of promoter-proximal poly(A) sites in introns or ORFs and increased utilization of promoter-distal polyadenylation sites in intergenic regions. This extensively affects gene expression beyond regulating mRNA abundance by changing mRNA length and by altering the configuration of open reading frames. Our study highlights the impact of post-transcriptional mechanisms on stress-dependent gene regulation and reveals the differential expression of alternatively polyadenylated transcripts as a common stress-induced mechanism in mammalian cells.

Improved binding site assignment by high-resolution mapping of RNA-protein interactions using iCLIP.

Individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP) allows the determination of crosslinking sites of RNA-binding proteins (RBPs) on RNAs. iCLIP is based on ultraviolet light crosslinking of RBPs to RNA, reverse transcription and high-throughput sequencing of fragments terminating at the site of crosslinking. As a result, start sites of iCLIP fragments are expected to cluster with a narrow distribution, typically representing the site of direct interaction between the RBP and the RNA. Here we show that for several RBPs (eIF4A3, PTB, SRSF3, SRSF4 and hnRNP L), the start sites of iCLIP fragments show a fragment length-dependent broader distribution that can be shifted to positions upstream of the known RNA-binding site. We developed an analysis tool that identifies these shifts and can improve the positioning of RBP binding sites.

mRNA 3'end processing: A tale of the tail reaches the clinic.

Recent advances reveal mRNA 3'end processing as a highly regulated process that fine-tunes posttranscriptional gene expression. This process can affect the site and/or the efficiency of 3'end processing, controlling the quality and the quantity of substrate mRNAs. The regulation of 3'end processing plays a central role in fundamental physiology such as blood coagulation and innate immunity. In addition, errors in mRNA 3'end processing have been associated with a broad spectrum of human diseases, including cancer. We summarize and discuss the paradigmatic shift in the understanding of 3'end processing as a mechanism of posttranscriptional gene regulation that has reached clinical medicine.