Translation and mRNA Stability Control.

Messenger RNA (mRNA) stability and translational efficiency are two crucial aspects of the post-transcriptional process that profoundly impact protein production in a cell. While it is widely known that ribosomes produce proteins, studies during the past decade have surprisingly revealed that ribosomes also control mRNA stability in a codon-dependent manner, a process referred to as codon optimality. Therefore, codons, the three-nucleotide words read by the ribosome, have a potent effect on mRNA stability and provide cis-regulatory information that extends beyond the amino acids they encode. While the codon optimality molecular mechanism is still unclear, the translation elongation rate appears to trigger mRNA decay. Thus, transfer RNAs emerge as potential master gene regulators affecting mRNA stability. Furthermore, while few factors related to codon optimality have been identified in yeast, the orthologous genes in vertebrates do not necessary share the same functions. Here, we discuss codon optimality findings and gene regulation layers related to codon composition in different eukaryotic species.

Mechanisms of readthrough mitigation reveal principles of GCN1-mediated translational quality control.

Readthrough into the 3' untranslated region (3' UTR) of the mRNA results in the production of aberrant proteins. Metazoans efficiently clear readthrough proteins, but the underlying mechanisms remain unknown. Here, we show in Caenorhabditis elegans and mammalian cells that readthrough proteins are targeted by a coupled, two-level quality control pathway involving the BAG6 chaperone complex and the ribosome-collision-sensing protein GCN1. Readthrough proteins with hydrophobic C-terminal extensions (CTEs) are recognized by SGTA-BAG6 and ubiquitylated by RNF126 for proteasomal degradation. Additionally, cotranslational mRNA decay initiated by GCN1 and CCR4/NOT limits the accumulation of readthrough products. Unexpectedly, selective ribosome profiling uncovered a general role of GCN1 in regulating translation dynamics when ribosomes collide at nonoptimal codons, enriched in 3' UTRs, transmembrane proteins, and collagens. GCN1 dysfunction increasingly perturbs these protein classes during aging, resulting in mRNA and proteome imbalance. Our results define GCN1 as a key factor acting during translation in maintaining protein homeostasis.

Following the Birth, Life, and Death of mRNAs in Single Cells.

Recent advances in single-molecule imaging of mRNAs in fixed and living cells have enabled the lives of mRNAs to be studied with unprecedented spatial and temporal detail. These approaches have moved beyond simply being able to observe specific events and have begun to allow an understanding of how regulation is coupled between steps in the mRNA life cycle. Additionally, these methodologies are now being applied in multicellular systems and animals to provide more nuanced insights into the physiological regulation of RNA metabolism.

Synonymous but Not Silent: The Codon Usage Code for Gene Expression and Protein Folding.

Codon usage bias, the preference for certain synonymous codons, is found in all genomes. Although synonymous mutations were previously thought to be silent, a large body of evidence has demonstrated that codon usage can play major roles in determining gene expression levels and protein structures. Codon usage influences translation elongation speed and regulates translation efficiency and accuracy. Adaptation of codon usage to tRNA expression determines the proteome landscape. In addition, codon usage biases result in nonuniform ribosome decoding rates on mRNAs, which in turn influence the cotranslational protein folding process that is critical for protein function in diverse biological processes. Conserved genome-wide correlations have also been found between codon usage and protein structures. Furthermore, codon usage is a major determinant of mRNA levels through translation-dependent effects on mRNA decay and translation-independent effects on transcriptional and posttranscriptional processes. Here, we discuss the multifaceted roles and mechanisms of codon usage in different gene regulatory processes.

Recording of elapsed time and temporal information about biological events using Cas9.

DNA has not been utilized to record temporal information, although DNA has been used to record biological information and to compute mathematical problems. Here, we found that indel generation by Cas9 and guide RNA can occur at steady rates, in contrast to typical dynamic biological reactions, and the accumulated indel frequency can be a function of time. By measuring indel frequencies, we developed a method for recording and measuring absolute time periods over hours to weeks in mammalian cells. These time-recordings were conducted in several cell types, with different promoters and delivery vectors for Cas9, and in both cultured cells and cells of living mice. As applications, we recorded the duration of chemical exposure and the lengths of elapsed time since the onset of biological events (e.g., heat exposure and inflammation). We propose that our systems could serve as synthetic "DNA clocks."

PNPT1 Release from Mitochondria during Apoptosis Triggers Decay of Poly(A) RNAs.

Widespread mRNA decay, an unappreciated feature of apoptosis, enhances cell death and depends on mitochondrial outer membrane permeabilization (MOMP), TUTases, and DIS3L2. Which RNAs are decayed and the decay-initiating event are unknown. Here, we show extensive decay of mRNAs and poly(A) noncoding (nc)RNAs at the 3' end, triggered by the mitochondrial intermembrane space 3'-to-5' exoribonuclease PNPT1, released during MOMP. PNPT1 knockdown inhibits apoptotic RNA decay and reduces apoptosis, while ectopic expression of PNPT1, but not an RNase-deficient mutant, increases RNA decay and cell death. The 3' end of PNPT1 substrates thread through a narrow channel. Many non-poly(A) ncRNAs contain 3'-secondary structures or bind proteins that may block PNPT1 activity. Indeed, mutations that disrupt the 3'-stem-loop of a decay-resistant ncRNA render the transcript susceptible, while adding a 3'-stem-loop to an mRNA prevents its decay. Thus, PNPT1 release from mitochondria during MOMP initiates apoptotic decay of RNAs lacking 3'-structures.

Post-transcriptional Regulation of De Novo Lipogenesis by mTORC1-S6K1-SRPK2 Signaling.

mTORC1 is a signal integrator and master regulator of cellular anabolic processes linked to cell growth and survival. Here, we demonstrate that mTORC1 promotes lipid biogenesis via SRPK2, a key regulator of RNA-binding SR proteins. mTORC1-activated S6K1 phosphorylates SRPK2 at Ser494, which primes Ser497 phosphorylation by CK1. These phosphorylation events promote SRPK2 nuclear translocation and phosphorylation of SR proteins. Genome-wide transcriptome analysis reveals that lipid biosynthetic enzymes are among the downstream targets of mTORC1-SRPK2 signaling. Mechanistically, SRPK2 promotes SR protein binding to U1-70K to induce splicing of lipogenic pre-mRNAs. Inhibition of this signaling pathway leads to intron retention of lipogenic genes, which triggers nonsense-mediated mRNA decay. Genetic or pharmacological inhibition of SRPK2 blunts de novo lipid synthesis, thereby suppressing cell growth. These results thus reveal a novel role of mTORC1-SRPK2 signaling in post-transcriptional regulation of lipid metabolism and demonstrate that SRPK2 is a potential therapeutic target for mTORC1-driven metabolic disorders.

Single-molecule imaging reveals translation-dependent destabilization of mRNAs.

The relationship between mRNA translation and decay is incompletely understood, with conflicting reports suggesting that translation can either promote decay or stabilize mRNAs. The effect of translation on mRNA decay has mainly been studied using ensemble measurements and global transcription and translation inhibitors, which can have pleiotropic effects. We developed a single-molecule imaging approach to control the translation of a specific transcript that enabled simultaneous measurement of translation and mRNA decay. Our results demonstrate that mRNA translation reduces mRNA stability, and mathematical modeling suggests that this process is dependent on ribosome flux. Furthermore, our results indicate that miRNAs mediate efficient degradation of both translating and non-translating target mRNAs and reveal a predominant role for mRNA degradation in miRNA-mediated regulation. Simultaneous observation of translation and decay of single mRNAs provides a framework to directly study how these processes are interconnected in cells.

Control of mRNA fate by its encoded nascent polypeptide.

Cells tightly regulate mRNA processing, localization, and stability to ensure accurate gene expression in diverse cellular states and conditions. Most of these regulatory steps have traditionally been thought to occur before translation by the action of RNA-binding proteins. Several recent discoveries highlight multiple co-translational mechanisms that modulate mRNA translation, localization, processing, and stability. These mechanisms operate by recognition of the nascent protein, which is necessarily coupled to its encoding mRNA during translation. Hence, the distinctive sequence or structure of a particular nascent chain can recruit recognition factors with privileged access to the corresponding mRNA in an otherwise crowded cellular environment. Here, we draw on both well-established and recent examples to provide a conceptual framework for how cells exploit nascent protein recognition to direct mRNA fate. These mechanisms allow cells to dynamically and specifically regulate their transcriptomes in response to changes in cellular states to maintain protein homeostasis.

PTBP1-activated co-transcriptional splicing controls epigenetic status of pluripotent stem cells.

Many spliceosomal introns are excised from nascent transcripts emerging from RNA polymerase II (RNA Pol II). The extent of cell-type-specific regulation and possible functions of such co-transcriptional events remain poorly understood. We examined the role of the RNA-binding protein PTBP1 in this process using an acute depletion approach followed by the analysis of chromatin- and RNA Pol II-associated transcripts. We show that PTBP1 activates the co-transcriptional excision of hundreds of introns, a surprising effect given that this protein is known to promote intron retention. Importantly, some co-transcriptionally activated introns fail to complete their splicing without PTBP1. In a striking example, retention of a PTBP1-dependent intron triggers nonsense-mediated decay of transcripts encoding DNA methyltransferase DNMT3B. We provide evidence that this regulation facilitates the natural decline in DNMT3B levels in developing neurons and protects differentiation-specific genes from ectopic methylation. Thus, PTBP1-activated co-transcriptional splicing is a widespread phenomenon mediating epigenetic control of cellular identity.

ARS2 instructs early transcription termination-coupled RNA decay by recruiting ZC3H4 to nascent transcripts.

The RNA-binding ARS2 protein is centrally involved in both early RNA polymerase II (RNAPII) transcription termination and transcript decay. Despite its essential nature, the mechanisms by which ARS2 enacts these functions have remained unclear. Here, we show that a conserved basic domain of ARS2 binds a corresponding acidic-rich, short linear motif (SLiM) in the transcription restriction factor ZC3H4. This interaction recruits ZC3H4 to chromatin to elicit RNAPII termination, independent of other early termination pathways defined by the cleavage and polyadenylation (CPA) and Integrator (INT) complexes. We find that ZC3H4, in turn, forms a direct connection to the nuclear exosome targeting (NEXT) complex, hereby facilitating rapid degradation of the nascent RNA. Hence, ARS2 instructs the coupled transcription termination and degradation of the transcript onto which it is bound. This contrasts with ARS2 function at CPA-instructed termination sites where the protein exclusively partakes in RNA suppression via post-transcriptional decay.

Targeted pseudouridylation: An approach for suppressing nonsense mutations in disease genes.

Nonsense mutations create premature termination codons (PTCs), activating the nonsense-mediated mRNA decay (NMD) pathway to degrade most PTC-containing mRNAs. The undegraded mRNA is translated, but translation terminates at the PTC, leading to no production of the full-length protein. This work presents targeted PTC pseudouridylation, an approach for nonsense suppression in human cells. Specifically, an artificial box H/ACA guide RNA designed to target the mRNA PTC can suppress both NMD and premature translation termination in various sequence contexts. Targeted pseudouridylation exhibits a level of suppression comparable with that of aminoglycoside antibiotic treatments. When targeted pseudouridylation is combined with antibiotic treatment, a much higher level of suppression is observed. Transfection of a disease model cell line (carrying a chromosomal PTC) with a designer guide RNA gene targeting the PTC also leads to nonsense suppression. Thus, targeted pseudouridylation is an RNA-directed gene-specific approach that suppresses NMD and concurrently promotes PTC readthrough.

Chromatin modifier HUSH co-operates with RNA decay factor NEXT to restrict transposable element expression.

Transposable elements (TEs) are widespread genetic parasites known to be kept under tight transcriptional control. Here, we describe a functional connection between the mouse-orthologous "nuclear exosome targeting" (NEXT) and "human silencing hub" (HUSH) complexes, involved in nuclear RNA decay and the epigenetic silencing of TEs, respectively. Knocking out the NEXT component ZCCHC8 in embryonic stem cells results in elevated TE RNA levels. We identify a physical interaction between ZCCHC8 and the MPP8 protein of HUSH and establish that HUSH recruits NEXT to chromatin at MPP8-bound TE loci. However, while NEXT and HUSH both dampen TE RNA expression, their activities predominantly affect shorter non-polyadenylated and full-length polyadenylated transcripts, respectively. Indeed, our data suggest that the repressive action of HUSH promotes a condition favoring NEXT RNA decay activity. In this way, transcriptional and post-transcriptional machineries synergize to suppress the genotoxic potential of TE RNAs.

DNA damage promotes HLA class I presentation by stimulating a pioneer round of translation-associated antigen production.

Antigen presentation by the human leukocyte antigen (HLA) on the cell surface is critical for the transduction of the immune signal toward cytotoxic T lymphocytes. DNA damage upregulates HLA class I presentation; however, the mechanism is unclear. Here, we show that DNA-damage-induced HLA (di-HLA) presentation requires an immunoproteasome, PSMB8/9/10, and antigen-transporter, TAP1/2, demonstrating that antigen production is essential. Furthermore, we show that di-HLA presentation requires ATR, AKT, mTORC1, and p70-S6K signaling. Notably, the depletion of CBP20, a factor initiating the pioneer round of translation (PRT) that precedes nonsense-mediated mRNA decay (NMD), abolishes di-HLA presentation, suggesting that di-antigen production requires PRT. RNA-seq analysis demonstrates that DNA damage reduces NMD transcripts in an ATR-dependent manner, consistent with the requirement for ATR in the initiation of PRT/NMD. Finally, bioinformatics analysis identifies that PRT-derived 9-mer peptides bind to HLA and are potentially immunogenic. Therefore, DNA damage signaling produces immunogenic antigens by utilizing the machinery of PRT/NMD.

Sensing of individual stalled 80S ribosomes by Fap1 for nonfunctional rRNA turnover.

Cells can respond to stalled ribosomes by sensing ribosome collisions and employing quality control pathways. How ribosome stalling is resolved without collisions, however, has remained elusive. Here, focusing on noncolliding stalling exhibited by decoding-defective ribosomes, we identified Fap1 as a stalling sensor triggering 18S nonfunctional rRNA decay via polyubiquitination of uS3. Ribosome profiling revealed an enrichment of Fap1 at the translation initiation site but also an association with elongating individual ribosomes. Cryo-EM structures of Fap1-bound ribosomes elucidated Fap1 probing the mRNA simultaneously at both the entry and exit channels suggesting an mRNA stasis sensing activity, and Fap1 sterically hinders the formation of canonical collided di-ribosomes. Our findings indicate that individual stalled ribosomes are the potential signal for ribosome dysfunction, leading to accelerated turnover of the ribosome itself.

AKT constitutes a signal-promoted alternative exon-junction complex that regulates nonsense-mediated mRNA decay.

Despite a long appreciation for the role of nonsense-mediated mRNA decay (NMD) in destroying faulty, disease-causing mRNAs and maintaining normal, physiologic mRNA abundance, additional effectors that regulate NMD activity in mammalian cells continue to be identified. Here, we describe a haploid-cell genetic screen for NMD effectors that has unexpectedly identified 13 proteins constituting the AKT signaling pathway. We show that AKT supersedes UPF2 in exon-junction complexes (EJCs) that are devoid of RNPS1 but contain CASC3, defining an unanticipated insulin-stimulated EJC. Without altering UPF1 RNA binding or ATPase activity, AKT-mediated phosphorylation of the UPF1 CH domain at T151 augments UPF1 helicase activity, which is critical for NMD and also decreases the dependence of helicase activity on ATP. We demonstrate that upregulation of AKT signaling contributes to the hyperactivation of NMD that typifies Fragile X syndrome, as exemplified using FMR1-KO neural stem cells derived from induced pluripotent stem cells.

Integrative omics indicate FMRP sequesters mRNA from translation and deadenylation in human neuronal cells.

How fragile X syndrome protein (FMRP) binds mRNAs and regulates mRNA metabolism remains unclear. Our previous work using human neuronal cells focused on mRNAs targeted for nonsense-mediated mRNA decay (NMD), which we showed are generally bound by FMRP and destabilized upon FMRP loss. Here, we identify >400 high-confidence FMRP-bound mRNAs, only ∼35% of which are NMD targets. Integrative transcriptomics together with SILAC-LC-MS/MS reveal that FMRP loss generally results in mRNA destabilization and more protein produced per FMRP target. We use our established RIP-seq technology to show that FMRP footprints are independent of protein-coding potential, target GC-rich and structured sequences, and are densest in 5' UTRs. Regardless of where within an mRNA FMRP binds, we find that FMRP protects mRNAs from deadenylation and directly binds the cytoplasmic poly(A)-binding protein. Our results reveal how FMRP sequesters polyadenylated mRNAs into stabilized and translationally repressed complexes, whose regulation is critical for neurogenesis and synaptic plasticity.

Minding the message: tactics controlling RNA decay, modification, and translation in virus-infected cells.

With their categorical requirement for host ribosomes to translate mRNA, viruses provide a wealth of genetically tractable models to investigate how gene expression is remodeled post-transcriptionally by infection-triggered biological stress. By co-opting and subverting cellular pathways that control mRNA decay, modification, and translation, the global landscape of post-transcriptional processes is swiftly reshaped by virus-encoded factors. Concurrent host cell-intrinsic countermeasures likewise conscript post-transcriptional strategies to mobilize critical innate immune defenses. Here we review strategies and mechanisms that control mRNA decay, modification, and translation in animal virus-infected cells. Besides settling infection outcomes, post-transcriptional gene regulation in virus-infected cells epitomizes fundamental physiological stress responses in health and disease.

Functional interaction between the RNA exosome and the sirtuin deacetylase Hst3 maintains transcriptional homeostasis.

Eukaryotic cells maintain an optimal level of mRNAs through unknown mechanisms that balance RNA synthesis and degradation. We found that inactivation of the RNA exosome leads to global reduction of nascent mRNA transcripts, and that this defect is accentuated by loss of deposition of histone variant H2A.Z. We identify the mRNA for the sirtuin deacetylase Hst3 as a key target for the RNA exosome that mediates communication between RNA degradation and transcription machineries. These findings reveal how the RNA exosome and H2A.Z function together to control a deacetylase, ensuring proper levels of transcription in response to changes in RNA degradation.

NMD is required for timely cell fate transitions by fine-tuning gene expression and regulating translation.

Cell fate transitions depend on balanced rewiring of transcription and translation programs to mediate ordered developmental progression. Components of the nonsense-mediated mRNA decay (NMD) pathway have been implicated in regulating embryonic stem cell (ESC) differentiation, but the exact mechanism is unclear. Here we show that NMD controls expression levels of the translation initiation factor Eif4a2 and its premature termination codon-encoding isoform (Eif4a2PTC ). NMD deficiency leads to translation of the truncated eIF4A2PTC protein. eIF4A2PTC elicits increased mTORC1 activity and translation rates and causes differentiation delays. This establishes a previously unknown feedback loop between NMD and translation initiation. Furthermore, our results show a clear hierarchy in the severity of target deregulation and differentiation phenotypes between NMD effector KOs (Smg5 KO > Smg6 KO > Smg7 KO), which highlights heterodimer-independent functions for SMG5 and SMG7. Together, our findings expose an intricate link between mRNA homeostasis and mTORC1 activity that must be maintained for normal dynamics of cell state transitions.