Multifunctional RNA-binding proteins influence mRNA abundance and translational efficiency of distinct sets of target genes.
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ABSTRACT:
SUBMITTER: Schneider-Lunitz V
PROVIDER: S-EPMC8687540 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Gene regulation via N6-methyladenosine (m6A) in mRNA involves RNA-binding proteins that recognize m6A via a YT521-B homology (YTH) domain. The plant YTH domain proteins ECT2 and ECT3 act genetically redundantly in stimulating cell proliferation during organogenesis, but several fundamental questions regarding their mode of action remain unclear. Here, we use HyperTRIBE (targets of RNA-binding proteins identified by editing) to show that most ECT2 and ECT3 targets overlap, with only a few examples of preferential targeting by either of the two proteins. HyperTRIBE in different mutant backgrounds also provides direct views of redundant, ectopic, and specific target interactions of the two proteins. We also show that contrary to conclusions of previous reports, ECT2 does not accumulate in the nucleus. Accordingly, inactivation of ECT2, ECT3, and their surrogate ECT4 does not change patterns of polyadenylation site choice in ECT2/3 target mRNAs, but does lead to lower steady-state accumulation of target mRNAs. In addition, mRNA and microRNA expression profiles show indications of stress response activation in ect2/ect3/ect4 mutants, likely via indirect effects. Thus, previous suggestions of control of alternative polyadenylation by ECT2 are not supported by evidence, and ECT2 and ECT3 act largely redundantly to regulate target mRNA, including its abundance, in the cytoplasm.
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Project description:BackgroundCDK9 is the catalytic subunit of the Positive Transcription Elongation Factor b (P-TEFb), which phosphorylates the CTD of RNAPII and negative elongation factors enabling for productive elongation after initiation. CDK9 associates with T-type cyclins and cyclin K and its activity is tightly regulated in cells at different levels. CDK9 is also the catalytic subunit of TAK (Tat activating Kinase), essential for HIV1 replication. Because of CDK9's potential as a therapeutic target in AIDS, cancer, inflammation, and cardiomyophathy it is important to understand the consequences of CDK9 inhibition. A previous gene expression profiling study performed with human glioblastoma T98G cells in which CDK9 activity was inhibited either with a dominant negative mutant form of CDK9 (dnCDK9) or the pharmacological inhibitor Flavopiridol unveiled striking differences in gene expression effects. In the present report we extended these studies by (1) using both immortalized normal human fibroblasts and primary human astrocytes, (2) eliminating potential experimental variability due to transduction methodology and (3) also modulating CDK9 activity with siRNA.FindingsStriking differences in the effects on gene expression resulting from the strategy used to inhibit CDK9 activity (dnCDK9 or FVP) remain even when potential variability due to viral transduction is eliminated. siRNA mediated CDK9 knockdown in human fibroblasts and astrocytes efficiently reduced CDK9 expression and led to potent changes in gene expression that exhibit little correlation with the effects of dnCDK9 or FVP. Interestingly, HEXIM1 a validated CDK9 target gene, was found to be potently downregulated by dnCDK9, FVP and siCDK9, but the cluster of genes with expression profiles similar to HEXIM1 was small. Finally, cluster analysis of all treatments revealed higher correlation between treatments than cell type origin.ConclusionThe nature of the strategy used to inhibit CDK9 profoundly affects the patterns of gene expression resulting from CDK9 inhibition. These results suggest multiple variables that affect outcome, including kinetics of inhibition, potency, off-target effects, and selectivity issues. This is particularly important when considering CDK9 as a potential target for therapeutic intervention.
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Project description:Post-transcriptional regulation by microRNAs and siRNAs depends not only on characteristics of individual binding sites in target mRNA molecules, but also on system-level properties such as overall molecular concentrations. We hypothesize that an intracellular pool of microRNAs/siRNAs faced with a larger number of available predicted target transcripts will downregulate each individual target gene to a lesser extent. To test this hypothesis, we analyzed mRNA expression change from 178 microRNA and siRNA transfection experiments in two cell lines. We find that downregulation of particular genes mediated by microRNAs and siRNAs indeed varies with the total concentration of available target transcripts. We conclude that to interpret and design experiments involving gene regulation by small RNAs, global properties, such as target mRNA abundance, need to be considered in addition to local determinants. We propose that analysis of microRNA/siRNA targeting would benefit from a more quantitative definition, rather than simple categorization of genes as 'target' or 'not a target.' Our results are important for understanding microRNA regulation and may also have implications for siRNA design and small RNA therapeutics.
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Project description:High gene expression of enzymes partaking in recombinant production pathways is a desirable trait among cell factories belonging to all different kingdoms of life. High enzyme abundance is generally aimed for by utilizing strong promoters, which ramp up gene transcription and mRNA levels. Increased protein abundance can alternatively be achieved by optimizing the expression on the post-transcriptional level. Here, we evaluated protein synthesis with a previously proposed optimized gene expression architecture, in which mRNA stability and translation initiation are modulated by genetic parts such as self-cleaving ribozymes and a bicistronic design, which have initially been described to support the standardization of gene expression. The optimized gene expression architecture was tested in Pseudomonas taiwanensis VLB120, a promising, novel microbial cell factory. The expression cassette was employed on a plasmid basis and after single genomic integration. We used three constitutive and two inducible promoters to drive the expression of two fluorescent reporter proteins and a short acetoin biosynthesis pathway. The performance was confronted with that of a traditional expression cassette harboring the same promoter and gene of interest but lacking the genetic parts for increased expression efficiency. The optimized expression cassette granted higher protein abundance independently of the expression basis or promoter used proving its value for applications requiring high protein abundance.
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Project description:Programmable protein scaffolds are invaluable in the development of genome engineering tools. The pentatricopeptide repeat (PPR) protein is an attractive platform for RNA manipulation because of its programmable RNA-binding selectivity, which is determined by the combination of amino acid species at three specific sites in the PPR motif. Translation is a key RNA regulatory step that determines the final gene expression level and is involved in various human diseases. In this study, designer PPR protein was used to develop a translational enhancement technique by fusion with the translation initiation factor eIF4G. The results showed that the PPR-eIF4G fusion protein could activate the translation of endogenous c-Myc and p53 mRNAs and control cell fate, indicating that PPR-based translational enhancement is a versatile technique applicable to various endogenous mRNAs in mammalian cells. In addition, the translational enhancement was dependent on both the target position and presence of eIF4G, suggesting the presence of an unknown translation activation mechanism.
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Project description:The modest correlation between mRNA expression and protein abundance in large-scale data sets is explained in part by experimental challenges, such as technological limitations, and in part by fundamental biological factors in the transcription and translation processes. Among various factors affecting the mRNA-protein correlation, the roles of biological factors related to translation are poorly understood. In this study, using experimental mRNA expression and protein abundance data collected from Desulfovibrio vulgaris by DNA microarray and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) proteomic analysis, we quantitatively examined the effects of several translational-efficiency-related sequence features on mRNA-protein correlation. Three classes of sequence features were investigated according to different translational stages: (i) initiation, Shine-Dalgarno sequences, start codon identity, and start codon context; (ii) elongation, codon usage and amino acid usage; and (iii) termination, stop codon identity and stop codon context. Surprisingly, although it is widely accepted that translation initiation is the rate-limiting step for translation, our results showed that the mRNA-protein correlation was affected the most by the features at elongation stages, i.e., codon usage and amino acid composition (5.3-15.7% and 5.8-11.9% of the total variation of mRNA-protein correlation, respectively), followed by stop codon context and the Shine-Dalgarno sequence (3.7-5.1% and 1.9-3.8%, respectively). Taken together, all sequence features contributed to 15.2-26.2% of the total variation of mRNA-protein correlation. This study provides the first comprehensive quantitative analysis of the mRNA-protein correlation in bacterial D. vulgaris and adds new insights into the relative importance of various sequence features in prokaryotic protein translation.
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Project description:Translation rate per mRNA molecule correlates positively with mRNA abundance. As a result, protein levels do not scale linearly with mRNA levels, but instead scale with the abundance of mRNA raised to the power of an 'amplification exponent'. Here we show that to quantitate translational control, the translation rate must be decomposed into two components. One, TRmD, depends on the mRNA level and defines the amplification exponent. The other, TRmIND, is independent of mRNA amount and impacts the correlation coefficient between protein and mRNA levels. We show that in Saccharomyces cerevisiae TRmD represents ?20% of the variance in translation and directs an amplification exponent of 1.20 with a 95% confidence interval [1.14, 1.26]. TRmIND constitutes the remaining ?80% of the variance in translation and explains ?5% of the variance in protein expression. We also find that TRmD and TRmIND are preferentially determined by different mRNA sequence features: TRmIND by the length of the open reading frame and TRmD both by a ?60 nucleotide element that spans the initiating AUG and by codon and amino acid frequency. Our work provides more appropriate estimates of translational control and implies that TRmIND is under different evolutionary selective pressures than TRmD.
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Project description:Catechol-O-methyltransferase (COMT) is a major enzyme controlling catecholamine levels that plays a central role in cognition, affective mood and pain perception. There are three common COMT haplotypes in the human population reported to have functional effects, divergent in two synonymous and one nonsynonymous position. We demonstrate that one of the haplotypes, carrying the non-synonymous variation known to code for a less stable protein, exhibits increased protein expression in vitro. This increased protein expression, which would compensate for lower protein stability, is solely produced by a synonymous variation (C(166)T) situated within the haplotype and located in the 5' region of the RNA transcript. Based on mRNA secondary structure predictions, we suggest that structural destabilization near the start codon caused by the T allele could be related to the observed increase in COMT expression. Our folding simulations of the tertiary mRNA structures demonstrate that destabilization by the T allele lowers the folding transition barrier, thus decreasing the probability of occupying its native state. These data suggest a novel structural mechanism whereby functional synonymous variations near the translation initiation codon affect the translation efficiency via entropy-driven changes in mRNA dynamics and present another example of stable compensatory genetic variations in the human population.
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Project description:The serotonin transporter (SERT, SLC6A4) is a Na+-dependent transporter that regulates the availability of serotonin (5-HT, 5-hydroxytryptamine), a key neurotransmitter and hormone in the brain and the intestine. The human SERT gene consists of two alternate promoters that drive expression of an identical SERT protein. However, there are different mRNA transcript variants derived from these two promoters that differ in their 5' untranslated region (5'UTR), which is the region of the mRNA upstream from the protein-coding region. Two of these transcripts contain exon-1a and are abundant in neuronal tissue, whereas the third transcript contains exon-1c and is abundant in the intestine. The 3'UTR is nearly identical among the transcripts. Current studies tested the hypothesis that the UTRs of SERT influence its expression in intestinal epithelial cells (IECs) by controlling mRNA or protein levels. The SERT UTRs were cloned into luciferase reporter plasmids and luciferase mRNA and activity were measured following transient transfection of the UTR constructs into the model IEC Caco-2. Luciferase activity and mRNA abundance were higher than the empty vector for two of the three 5'UTR variants. Calculation of translation index (luciferase activity divided by the relative luciferase mRNA level) revealed that the exon-1a containing 5'UTRs had enhanced translation when compared to the exon-1c containing 5'UTR which exhibited a low translation efficiency. Compared to the empty vector, the SERT 3'UTR markedly decreased luciferase activity. In silico analysis of the SERT 3'UTR revealed many conserved potential miRNA binding sites that may be responsible for this decrease. In conclusion, we have shown that the UTRs of SERT regulate mRNA abundance and protein expression. Delineating the molecular basis by which the UTRs of SERT influence its expression will lead to an increased understanding of post-transcriptional regulation of SERT in GI disorders associated with altered 5-HT availability.
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Project description:Bacterial translation is thought to initiate by base pairing of the 16S rRNA and the Shine-Dalgarno sequence in the mRNA's 5' untranslated region (UTR). However, transcriptomics has revealed that leaderless mRNAs, which completely lack any 5' UTR, are broadly distributed across bacteria and can initiate translation in the absence of the Shine-Dalgarno sequence. To investigate the mechanism of leaderless mRNA translation initiation, synthetic in vivo translation reporters were designed that systematically tested the effects of start codon accessibility, leader length, and start codon identity on leaderless mRNA translation initiation. Using these data, a simple computational model was built based on the combinatorial relationship of these mRNA features that can accurately classify leaderless mRNAs and predict the translation initiation efficiency of leaderless mRNAs. Thus, start codon accessibility, leader length, and start codon identity combine to define leaderless mRNA translation initiation in bacteria.
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