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.

Transcription feedback dynamics in the wake of cytoplasmic degradation shutdown

In the last decade, multiple studies have shown that cells maintain a balance of mRNA production and degradation in different settings, but the mechanisms by which cells implement this balance remain poorly understood. Here, we monitored cells’ mRNA and nascent mRNA profiles immediately following an acute depletion of Xrn1 - the main 5’-3’ mRNA exonuclease - that was previously implicated in balancing mRNA levels. We captured the detailed dynamics of the cells’ adaptation to rapid degradation of Xrn1 and observed a significant accumulation of mRNA, followed by global reduction in nascent transcription and a return to baseline mRNA levels. We present evidence that this transcriptional response is linked to cell cycle progression, and that it is not unique to Xrn1 depletion; rather, it is induced earlier when upstream factors in the 5’-3’ degradation pathway are perturbed. Using the detailed dynamic measurements we hypothesize a cell-cycle-linked feedback mechanism that monitors the accumulation of inputs to the 5’-3’ exonucleolytic pathway rather than its outputs.

Accurate inference of stochastic gene expression from nascent transcript heterogeneity

Transcriptional rates are often estimated by fitting the distribution of mature mRNA numbers measured using smFISH (single molecule fluorescence in situ hybridization) with the distribution predicted by the telegraph model of gene expression, which defines two promoter states of activity and inactivity. However, fluctuations in mature mRNA numbers are strongly affected by processes downstream of transcription. In addition, the telegraph model assumes one gene copy, but in experiments cells may have two gene copies as cells replicate their genome during the cell cycle. It is thus unclear how accurately the inferred parameters reflect transcription. To address these issues, here we measure both mature and nascent mRNA distributions of GAL10 in yeast cells using smFISH and classify each cell according to its cell cycle stage. We infer transcriptional parameters from mature and nascent mRNA distributions, with and without accounting for cell cycle stage and compare the results to live-cell transcription measurements of the same gene. We conclude that: (i) not accounting for cell cycle dynamics in nascent mRNA data overestimates the magnitude of promoter switching rates and the initiation rate, and underestimates the fraction of time spent in the active state and the burst size. (ii) use of mature mRNA data, instead of nascent data, significantly increases the errors in parameter estimation and can mistakenly classify a gene as non-bursting. Furthermore, we show how to correctly adjust for measurement noise in smFISH at low nascent transcript numbers. Simulations with parameters estimated from nascent smFISH data corrected for cell cycle phases and measurement noise leads to autocorrelation functions that agree with those obtained from live-cell imaging. Therefore, our novel data curation method yields a quantitatively accurate picture of gene expression.

Nucleocytoplasmic shuttling of Gle1 impacts DDX1 at transcription termination sites

We uncovered a nuclear role for human Gle1 in coordinating transcription termination. When nucleocytoplasmic shuttling of Gle1 is disrupted, nascent mRNA transcripts are elongated. Gle1 colocalizes with DDX1, and loss of Gle1 shuttling impairs recruitment of DDX1 to CstF-64 and transcription termination foci, leading to improper pre-mRNA cleavage.

Analytical distributions for detailed models of stochastic gene expression in eukaryotic cells

The stochasticity of gene expression presents significant challenges to the modeling of genetic networks. A two-state model describing promoter switching, transcription, and messenger RNA (mRNA) decay is the standard model of stochastic mRNA dynamics in eukaryotic cells. Here, we extend this model to include mRNA maturation, cell division, gene replication, dosage compensation, and growth-dependent transcription. We derive expressions for the time-dependent distributions of nascent mRNA and mature mRNA numbers, provided two assumptions hold: 1) nascent mRNA dynamics are much faster than those of mature mRNA; and 2) gene-inactivation events occur far more frequently than gene-activation events. We confirm that thousands of eukaryotic genes satisfy these assumptions by using data from yeast, mouse, and human cells. We use the expressions to perform a sensitivity analysis of the coefficient of variation of mRNA fluctuations averaged over the cell cycle, for a large number of genes in mouse embryonic stem cells, identifying degradation and gene-activation rates as the most sensitive parameters. Furthermore, it is shown that, despite the model’s complexity, the time-dependent distributions predicted by our model are generally well approximated by the negative binomial distribution. Finally, we extend our model to include translation, protein decay, and auto-regulatory feedback, and derive expressions for the approximate time-dependent protein-number distributions, assuming slow protein decay. Our expressions enable us to study how complex biological processes contribute to the fluctuations of gene products in eukaryotic cells, as well as allowing a detailed quantitative comparison with experimental data via maximum-likelihood methods.

MicroRNAs Cause Accelerated Decay of Short-Tailed Target mRNAs

SummaryMicroRNAs (miRNAs) specify recruitment of deadenylases to mRNA targets. Despite this recruitment, we find that miRNAs have almost no effect on steady-state poly(A)-tail lengths of their targets in mouse fibroblasts, which motivates acquisition of pre-steady-state measurements of the effects of miRNAs on tail lengths, mRNA levels, and translational efficiencies. Effects on translational efficiency are minimal compared to effects on mRNA levels—even for newly transcribed target mRNAs. Effects on target mRNA levels accumulate as the mRNA population approaches steady state, whereas effects on tail lengths peak for recently transcribed target mRNAs and then subside. Computational modeling of this phenomenon reveals that miRNAs cause not only accelerated deadenylation of their targets but also accelerated decay of short-tailed target molecules. This unanticipated effect of miRNAs largely prevents short-tailed target mRNAs from accumulating despite accelerated target deadenylation. The net result is a nearly imperceptible change to the steady-state tail-length distribution of targeted mRNAs.HighlightsmiRNAs cause accelerated decay of short-tailed target moleculesThis accelerated decay largely prevents accumulation of short-tailed target mRNAsmiRNAs are similarly effective on short-lived and long-lived target mRNAsIn 3T3 cells, miRNA effects on translation are negligible—even for nascent mRNA

Conformational Dynamics of the RNA G-Quadruplex and its Effect on Translation Efficiency

During translation, intracellular mRNA folds co-transcriptionally and must refold following the passage of ribosome. The mRNAs can be entrapped in metastable structures during these folding events. In the present study, we evaluated the conformational dynamics of the kinetically favored, metastable, and hairpin-like structure, which disturbs the thermodynamically favored G-quadruplex structure, and its effect on co-transcriptional translation in prokaryotic cells. We found that nascent mRNA forms a metastable hairpin-like structure during co-transcriptional folding instead of the G-quadruplex structure. When the translation progressed co-transcriptionally before the metastable hairpin-like structure transition to the G-quadruplex, function of the G-quadruplex as a roadblock of the ribosome was sequestered. This suggested that kinetically formed RNA structures had a dominant effect on gene expression in prokaryotes. The results of this study indicate that it is critical to consider the conformational dynamics of RNA-folding to understand the contributions of the mRNA structures in controlling gene expression.

Distinct Functions of the Cap-Binding Complex in Stimulation of Nuclear mRNA Export

ABSTRACTCap-binding complex (CBC) associates cotranscriptionally with the cap structure at the 5′ end of nascent mRNA to protect it from exonucleolytic degradation. Here, we show that CBC promotes the targeting of an mRNA export adaptor, Yra1 (forming transcription export [TREX] complex with THO and Sub2), to the active genes and enhances mRNA export inSaccharomyces cerevisiae. Likewise, recruitment of Npl3 (an hnRNP involved in mRNA export via formation of export-competent ribonuclear protein complex [RNP]) to the active genes is facilitated by CBC. Thus, CBC enhances targeting of the export factors and promotes mRNA export. Such function of CBC is not mediated via THO and Sub2 of TREX, cleavage and polyadenylation factors, or Sus1 (that regulates mRNA export via transcription export 2 [TREX-2]). However, CBC promotes splicing ofSUS1mRNA and, consequently, Sus1 protein level and mRNA export via TREX-2. Collectively, our results support the hypothesis that CBC promotes recruitment of Yra1 and Npl3 to the active genes, independently of THO, Sub2, or cleavage and polyadenylation factors, and enhances mRNA export via TREX and RNP, respectively, in addition to its role in facilitatingSUS1mRNA splicing to increase mRNA export through TREX-2, revealing distinct stimulatory functions of CBC in mRNA export.

Measures of single- versus multiple-round translation argue against a mechanism to ensure coupling of transcription and translation

In prokaryotes, the synthesis of RNA and protein occurs simultaneously in the cytoplasm. A number of studies indicate that translation can strongly impact transcription, a phenomenon often attributed to physical coupling between RNA polymerase (RNAP) and the lead ribosome on the nascent mRNA. Whether there generally exists a mechanism to ensure or promote RNAP–ribosome coupling remains unclear. Here, we used an efficient hammerhead ribozyme and developed a reporter system to measure single- versus multiple-round translation in Escherichia coli. Six pairs of cotranscribed and differentially translated genes were analyzed. For five of them, the stoichiometry of the two protein products came no closer to unity (1:1) when the rounds of translation were severely reduced in wild-type cells. Introduction of mutation rpoB(I572N), which slows RNAP elongation, could promote coupling, as indicated by stoichiometric SspA and SspB products in the single-round assay. These data are consistent with models of stochastic coupling in which the probability of coupling depends on the relative rates of transcription and translation and suggest that RNAP often transcribes without a linked ribosome.