scholarly journals Seeking a Role for Translational Control by Alternative Polyadenylation in Saccharomyces cerevisiae

2021 ◽  
Vol 9 (9) ◽  
pp. 1885
Author(s):  
Rachael E. Turner ◽  
Traude H. Beilharz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Translational Control ◽  
Model Organism ◽  
Alternative Polyadenylation ◽  
Ribosome Profiling ◽  
Translation Efficiency ◽  
Mrna Isoforms ◽  
Cleavage And Polyadenylation ◽  
Made In

Alternative polyadenylation (APA) represents an important mechanism for regulating isoform-specific translation efficiency, stability, and localisation. Though some progress has been made in understanding its consequences in metazoans, the role of APA in the model organism Saccharomyces cerevisiae remains a relative mystery because, despite abundant studies on the translational state of mRNA, none differentiate mRNA isoforms’ alternative 3′-end. This review discusses the implications of alternative polyadenylation in S. cerevisiae using other organisms to draw inferences. Given the foundational role that research in this yeast has played in the discovery of the mechanisms of cleavage and polyadenylation and in the drivers of APA, it is surprising that such an inference is required. However, because advances in ribosome profiling are insensitive to APA, how it impacts translation is still unclear. To bridge the gap between widespread observed APA and the discovery of any functional consequence, we also provide a review of the experimental techniques used to uncover the functional importance of 3′ UTR isoforms on translation.

scholarly journals The RS Domain of Human CFIm68 Plays a Key Role in Selection Between Alternative Sites of Pre-mRNA Cleavage and Polyadenylation

2017 ◽  
Author(s):  
Jessica G. Hardy ◽  
Michael Tellier ◽  
Shona Murphy ◽  
Chris J. Norbury
Keyword(s):  
Molecular Mechanisms ◽  
Alternative Polyadenylation ◽  
Hek293 Cells ◽  
Translation Efficiency ◽  
Mrna Isoforms ◽  
Protein Coding ◽  
Mrna Cleavage ◽  
Cleavage And Polyadenylation ◽  
Alternative Sites

AbstractMany eukaryotic protein-coding genes give rise to alternative mRNA isoforms with identical protein-coding capacities but which differ in the extents of their 3´ untranslated regions (3´UTRs), due to the usage of alternative sites of pre-mRNA cleavage and polyadenylation. By governing the presence of regulatory 3´UTR sequences, this type of alternative polyadenylation (APA) can significantly influence the stability, localisation and translation efficiency of mRNA. Though a variety of molecular mechanisms for APA have been proposed, previous studies have identified a pivotal role for the multi-subunit cleavage factor I (CFIm) in this process in mammals. Here we show that, in line with previous reports, depletion of the CFIm 68 kDa subunit (CFIm68) by CRISPR/Cas9-mediated gene disruption in HEK293 cells leads to a shift towards the use of promoter-proximal poly(A) sites. Using these cells as the basis for a complementation assay, we show that CFIm68 lacking its arginine/serine-rich (RS) domain retains the ability to form a nuclear complex with other CFIm subunits, but selectively lacks the capacity to restore polyadenylation at promoter-distal sites. In addition, nanoparticle-mediated analysis indicates that the RS domain is extensively phosphorylated in vivo. Overall, these results suggest that the CFIm68 RS domain makes a key regulatory contribution to APA.

scholarly journals Loss of Ftsj1 perturbs codon-specific translation efficiency in the brain and is associated with X-linked intellectual disability

2021 ◽  
Vol 7 (13) ◽  
pp. eabf3072
Author(s):  
Y. Nagayoshi ◽  
T. Chujo ◽  
S. Hirata ◽  
H. Nakatsuka ◽  
C.-W. Chen ◽  
...  
Keyword(s):  
Intellectual Disability ◽  
State Level ◽  
Memory Deficits ◽  
Ribosome Profiling ◽  
Translation Efficiency ◽  
Trna Modification ◽  
Synaptic Organization ◽  
Transfer Rnas ◽  
The Brain

FtsJ RNA 2′-O-methyltransferase 1 (FTSJ1) gene has been implicated in X-linked intellectual disability (XLID), but the molecular pathogenesis is unknown. We show that Ftsj1 is responsible for 2′-O-methylation of 11 species of cytosolic transfer RNAs (tRNAs) at the anticodon region, and these modifications are abolished in Ftsj1 knockout (KO) mice and XLID patient–derived cells. Loss of 2′-O-methylation in Ftsj1 KO mouse selectively reduced the steady-state level of tRNAPhe in the brain, resulting in a slow decoding at Phe codons. Ribosome profiling showed that translation efficiency is significantly reduced in a subset of genes that need to be efficiently translated to support synaptic organization and functions. Ftsj1 KO mice display immature synaptic morphology and aberrant synaptic plasticity, which are associated with anxiety-like and memory deficits. The data illuminate a fundamental role of tRNA modification in the brain through regulation of translation efficiency and provide mechanistic insights into FTSJ1-related XLID.

scholarly journals Regulation, diversity and function of MECP2 exon and 3′UTR isoforms

2020 ◽  
Vol 29 (R1) ◽  
pp. R89-R99
Author(s):  
Deivid Carvalho Rodrigues ◽  
Marat Mufteev ◽  
James Ellis
Keyword(s):  
Dna Binding ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Alternative Polyadenylation ◽  
Cell Types ◽  
Protein Isoforms ◽  
Translation Efficiency ◽  
Mrna Isoforms ◽  
Exon 2 ◽  
Messenger Ribonucleic Acid

Abstract The methyl-CpG-binding protein 2 (MECP2) is a critical global regulator of gene expression. Mutations in MECP2 cause neurodevelopmental disorders including Rett syndrome (RTT). MECP2 exon 2 is spliced into two alternative messenger ribonucleic acid (mRNA) isoforms encoding MECP2-E1 or MECP2-E2 protein isoforms that differ in their N-termini. MECP2-E2, isolated first, was used to define the general roles of MECP2 in methyl-deoxyribonucleic acid (DNA) binding, targeting of transcriptional regulatory complexes, and its disease-causing impact in RTT. It was later found that MECP2-E1 is the most abundant isoform in the brain and its exon 1 is also mutated in RTT. MECP2 transcripts undergo alternative polyadenylation generating mRNAs with four possible 3′untranslated region (UTR) lengths ranging from 130 to 8600 nt. Together, the exon and 3′UTR isoforms display remarkable abundance disparity across cell types and tissues during development. These findings indicate discrete means of regulation and suggest that protein isoforms perform non-overlapping roles. Multiple regulatory programs have been explored to explain these disparities. DNA methylation patterns of the MECP2 promoter and first intron impact MECP2-E1 and E2 isoform levels. Networks of microRNAs and RNA-binding proteins also post-transcriptionally regulate the stability and translation efficiency of MECP2 3′UTR isoforms. Finally, distinctions in biophysical properties in the N-termini between MECP2-E1 and E2 lead to variable protein stabilities and DNA binding dynamics. This review describes the steps taken from the discovery of MECP2, the description of its key functions, and its association with RTT, to the emergence of evidence revealing how MECP2 isoforms are differentially regulated at the transcriptional, post-transcriptional and post-translational levels.

scholarly journals Dihydropteroate Synthase Mutations in Pneumocystis jiroveci Can Affect Sulfamethoxazole Resistance in a Saccharomyces cerevisiae Model

2004 ◽  
Vol 48 (7) ◽  
pp. 2617-2623 ◽  
Author(s):  
Peter Iliades ◽  
Steven R. Meshnick ◽  
Ian G. Macreadie
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Drug Resistance ◽  
Model Organism ◽  
Acid Synthesis ◽  
Aminobenzoic Acid ◽  
Pneumocystis Jiroveci ◽  
Sulfa Drug ◽  
Dihydropteroate Synthase ◽  
Dihydroneopterin Aldolase ◽  
Made In

ABSTRACT Dihydropteroate synthase (DHPS) mutations in Pneumocystis jiroveci have been associated epidemiologically with resistance to sulfamethoxazole (SMX). Since P. jiroveci cannot be cultured, inherent drug resistance cannot be measured. This study explores the effects of these mutations in a tractable model organism, Saccharomyces cerevisiae. Based on the sequence conservation between the DHPS enzymes of P. jiroveci and S. cerevisiae, together with the structural conservation of the three known DHPS structures, DHPS substitutions commonly observed in P. jiroveci were reverse engineered into the S. cerevisiae DHPS. Those mutations, T597A and P599S, can occur singly but are most commonly found together and are associated with SMX treatment failure. Mutations encoding the corresponding changes in the S. cerevisiae dhps were made in a yeast centromere vector, p414FYC, which encodes the native yeast DHPS as part of a trifunctional protein that also includes the two enzymes upstream of DHPS in the folic acid synthesis pathway, dihydroneopterin aldolase and 2-amino-4-hydroxymethyl dihydropteridine pyrophosphokinase. A yeast strain with dhps deleted was employed as the host strain, and transformants having DHPS activity were recovered. Mutants having both T597 and P599 substitutions had a requirement for p-aminobenzoic acid (PABA), consistent with resistance being associated with altered substrate binding. These mutants could be adapted for growth in the absence of PABA, which coincided with increased sulfa drug resistance. Upregulated PABA synthesis was thus implicated as a mechanism for sulfa drug resistance for mutants having two DHPS substitutions.

scholarly journals Translational control of ERK signaling through miRNA/4EHP-directed silencing

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Seyed Mehdi Jafarnejad ◽  
Clément Chapat ◽  
Edna Matta-Camacho ◽  
Idit Anna Gelbart ◽  
Geoffrey G Hesketh ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Translational Control ◽  
Mammalian Cells ◽  
Ribosome Profiling ◽  
Erk Signaling ◽  
Translational Repression ◽  
Signaling Cascade ◽  
Critical Function ◽  
Integral Role

MicroRNAs (miRNAs) exert a broad influence over gene expression by directing effector activities that impinge on translation and stability of mRNAs. We recently discovered that the cap-binding protein 4EHP is a key component of the mammalian miRNA-Induced Silencing Complex (miRISC), which mediates gene silencing. However, little is known about the mRNA repertoire that is controlled by the 4EHP/miRNA mechanism or its biological importance. Here, using ribosome profiling, we identify a subset of mRNAs that are translationally controlled by 4EHP. We show that the Dusp6 mRNA, which encodes an ERK1/2 phosphatase, is translationally repressed by 4EHP and a specific miRNA, miR-145. This promotes ERK1/2 phosphorylation, resulting in augmented cell growth and reduced apoptosis. Our findings thus empirically define the integral role of translational repression in miRNA-induced gene silencing and reveal a critical function for this process in the control of the ERK signaling cascade in mammalian cells.

scholarly journals mTOR-driven widespread exon skipping renders multifaceted gene regulation and proteome complexity

2020 ◽  
Author(s):  
Sze Cheng ◽  
Naima Ahmed Fahmi ◽  
Meeyeon Park ◽  
Jae-Woong Chang ◽  
Jiao Sun ◽  
...  
Keyword(s):  
Rna Processing ◽  
Mammalian Target Of Rapamycin ◽  
Exon Skipping ◽  
Alternative Polyadenylation ◽  
Translation Efficiency ◽  
Post Translational Modifications ◽  
Coding Regions ◽  
Protein Length ◽  
Processing Factors

AbstractThe mammalian target of rapamycin (mTOR) pathway is crucial in cell proliferation. Previously, we reported transcriptome-wide 3’-untranslated region (UTR) shortening by alternative polyadenylation upon mTOR activation and its impact on the proteome. Here, we further interrogated the mTOR-activated transcriptome and found that hyperactivation of mTOR promotes transcriptome-wide exon-skipping/exclusion, producing short isoform transcripts from genes. This widespread exon skipping confers multifarious regulations in the mTOR-controlled functional proteomics: alternative splicing (AS) in the 5’-UTR controls translation efficiency while AS in coding regions widely affects the protein length and functional domains. They also alter the half-life of proteins and affect the regulatory post-translational modifications. Among the RNA processing factors differentially regulated by mTOR signaling, we found that SRSF3 mechanistically facilitates exon skipping in the mTOR-activated transcriptome. This study reveals a role of mTOR in AS regulation and demonstrates that widespread AS is a multifaceted modulator of the mTOR-regulated functional proteome.

An mRNA processing pathway suppresses metastasis by governing translational control from the nucleus

2021 ◽  
Author(s):  
Albertas Navickas ◽  
Hosseinali Asgharian ◽  
Juliane Winkler ◽  
Lisa Fish ◽  
Kristle Garcia ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Cancer Progression ◽  
Translational Control ◽  
Gene Networks ◽  
Breast Cancer Cells ◽  
Metastatic Breast ◽  
Alternative Polyadenylation ◽  
Ribosome Profiling ◽  
Transcriptional Regulatory Mechanisms

Cancer cells often co-opt post-transcriptional regulatory mechanisms to achieve pathologic expression of gene networks that drive metastasis. Translational control is a major regulatory hub in oncogenesis, however its effects on cancer progression remain poorly understood. To address this, we used ribosome profiling to compare genome-wide translation efficiencies of poorly and highly metastatic breast cancer cells and patient-derived xenografts. We developed novel regression-based methods to analyze ribosome profiling and alternative polyadenylation data, and identified HNRNPC as a translational controller of a specific mRNA regulon. Mechanistically, HNRNPC, in concert with PABPC4, binds near to poly(A) signals, thereby governing the alternative polyadenylation of a set of mRNAs. We found that HNRNPC and PABPC4 are downregulated in highly metastatic cells, which causes HNRNPC-bound mRNAs to undergo 3' UTR lengthening and subsequently, translational repression. We showed that modulating HNRNPC expression impacts the metastatic capacity of breast cancer cells in xenograft mouse models. We also found that a small molecule, previously shown to induce a distal-to-proximal poly(A) site switching, counteracts the HNRNPC-PABPC4 driven deregulation of alternative polyadenylation and decreases the metastatic lung colonization by breast cancer cells in vivo.

scholarly journals Poly(A)-Binding Protein Acts in Translation Termination via Eukaryotic Release Factor 3 Interaction and Does Not Influence [PSI+] Propagation

2002 ◽  
Vol 22 (10) ◽  
pp. 3301-3315 ◽  
Author(s):  
Bertrand Cosson ◽  
Anne Couturier ◽  
Svetlana Chabelskaya ◽  
Denis Kiktev ◽  
Sergey Inge-Vechtomov ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Translational Control ◽  
Genetic Interaction ◽  
Translation Termination ◽  
Hybrid Approach ◽  
Translation Efficiency ◽  
Release Factor ◽  
Initiation Of Translation ◽  
Two Hybrid ◽  
Lines Of Evidence

ABSTRACT Recent studies of translational control suggest that translation termination may not be simply the end of synthesizing a protein but rather be involved in modulating both the translation efficiency and stability of a given transcript. Using recombinant eukaryotic release factor 3 (eRF3) and cellular extracts, we have shown for Saccharomyces cerevisiae that yeast eRF3 and Pab1p can interact. This interaction, mediated by the N+M domain of eRF3 and amino acids 473 to 577 of Pab1p, was demonstrated to be direct by the two-hybrid approach. We confirmed that a genetic interaction exists between eRF3 and Pab1p and showed that Pab1p overexpression enhances the efficiency of termination in SUP35 (eRF3) mutant and [PSI +] cells. This effect requires the interaction of Pab1p with eRF3. These data further strengthen the possibility that Pab1p has a role in coupling translation termination events with initiation of translation. Several lines of evidence indicate that Pab1p does not influence [PSI +] propagation. First, “[PSI +]-no-more” mutations do not affect eRF3-Pab1p two-hybrid interaction. Second, overexpression of PAB1 does not cure the [PSI +] phenotype or solubilize detectable amounts of eRF3. Third, prion-curing properties of overexpressed HSP104p, which is required for formation and maintenance of [PSI +], were not modified by excess Pab1p.

scholarly journals Deficiency of mitoribosomal S10 protein affects translation and splicing in Arabidopsis mitochondria

2019 ◽  
Author(s):  
Malgorzata Kwasniak-Owczarek ◽  
Urszula Kazmierczak ◽  
Artur Tomal ◽  
Pawel Mackiewicz ◽  
Hanna Janska
Keyword(s):  
Protein Synthesis ◽  
Translational Control ◽  
Ribosomal Proteins ◽  
Regulatory Element ◽  
Mitochondrial Gene ◽  
Ribosome Profiling ◽  
Translation Efficiency ◽  
Wild Type ◽  
Gene Encoding ◽  
Related Proteins

Abstract The ribosome is not only a protein-making machine, but also a regulatory element in protein synthesis. This view is supported by our earlier data showing that Arabidopsis mitoribosomes altered due to the silencing of the nuclear RPS10 gene encoding mitochondrial ribosomal protein S10 differentially translate mitochondrial transcripts compared with the wild-type. Here, we used ribosome profiling to determine the contribution of transcriptional and translational control in the regulation of protein synthesis in rps10 mitochondria compared with the wild-type ones. Oxidative phosphorylation system proteins are preferentially synthesized in wild-type mitochondria but this feature is lost in the mutant. The rps10 mitoribosomes show slightly reduced translation efficiency of most respiration-related proteins and at the same time markedly more efficiently synthesize ribosomal proteins and MatR and TatC proteins. The mitoribosomes deficient in S10 protein protect shorter transcript fragments which exhibit a weaker 3-nt periodicity compared with the wild-type. The decrease in the triplet periodicity is particularly drastic for genes containing introns. Notably, splicing is considerably less effective in the mutant, indicating an unexpected link between the deficiency of S10 and mitochondrial splicing. Thus, a shortage of the mitoribosomal S10 protein has wide-ranging consequences on mitochondrial gene expression.

scholarly journals Emerging Roles for 3′ UTRs in Neurons

2020 ◽  
Vol 21 (10) ◽  
pp. 3413
Author(s):  
Bongmin Bae ◽  
Pedro Miura
Keyword(s):  
Translational Control ◽  
Transcriptional Control ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Alternative Polyadenylation ◽  
Mrna Localization ◽  
Structural Features ◽  
Mrna Isoforms ◽  
Rna Sequences ◽  
Neuronal Functions

The 3′ untranslated regions (3′ UTRs) of mRNAs serve as hubs for post-transcriptional control as the targets of microRNAs (miRNAs) and RNA-binding proteins (RBPs). Sequences in 3′ UTRs confer alterations in mRNA stability, direct mRNA localization to subcellular regions, and impart translational control. Thousands of mRNAs are localized to subcellular compartments in neurons—including axons, dendrites, and synapses—where they are thought to undergo local translation. Despite an established role for 3′ UTR sequences in imparting mRNA localization in neurons, the specific RNA sequences and structural features at play remain poorly understood. The nervous system selectively expresses longer 3′ UTR isoforms via alternative polyadenylation (APA). The regulation of APA in neurons and the neuronal functions of longer 3′ UTR mRNA isoforms are starting to be uncovered. Surprising roles for 3′ UTRs are emerging beyond the regulation of protein synthesis and include roles as RBP delivery scaffolds and regulators of alternative splicing. Evidence is also emerging that 3′ UTRs can be cleaved, leading to stable, isolated 3′ UTR fragments which are of unknown function. Mutations in 3′ UTRs are implicated in several neurological disorders—more studies are needed to uncover how these mutations impact gene regulation and what is their relationship to disease severity.

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