Determinant Of mRNA Stability Research Articles

Distinct hypoxia-induced translational profiles of embryonic and adult-derived macrophages

Tissue resident macrophages are largely of embryonic (fetal liver) origin and long-lived, while bone marrow-derived macrophages (BMDM) are recruited following an acute perturbation, such as hypoxia in the setting of myocardial ischemia. Prior transcriptome analyses identified BMDM and fetal liver-derived macrophage (FLDM) differences at the RNA expression level. Posttranscriptional regulation determining mRNA stability and translation rate may override transcriptional signals in response to hypoxia. We profiled differentially regulated BMDM and FLDM transcripts in response to hypoxia at the level of mRNA translation. Using a translating ribosome affinity purification (TRAP) assay and RNA-seq, we identified non-overlapping transcripts with increased translation rate in BMDM (Ly6e, vimentin, PF4) and FLDM (Ccl7, Ccl2) after hypoxia. We further identified hypoxia-induced transcripts within these subsets that are regulated by the RNA-binding protein HuR. These findings define translational differences in macrophage subset gene expression programs, highlighting potential therapeutic targets in ischemic myocardium.

Molecular Mechanism of Long Noncoding RNA SNHG14 in Osteogenic Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells through the NEDD4L/FOXA2/PCP4 Axis.

Bone marrow-derived mesenchymal stem cells (BMSCs) have a superior potential of osteogenic differentiation (OD) and a promising stem cell type to treat bone defects. This study sought to investigate the molecular mechanism of long noncoding RNA small nucleolar RNA host gene 14 (SNHG14) in OD of BMSCs. Western blot analysis or RT-qPCR showed that SNHG14, neural precursor cell expressed developmentally downregulated 4-like (NEDD4L), and Purkinje cell protein 4 (PCP4) were upregulated whereas forkhead box A2 (FOXA2) was declined in OD of BMSCs. RT-qPCR and cell staining showed that SNHG14 downregulation repressed OD of BMSCs, as manifested by reductions in osteopontin and osteocalcin levels, the mineralization degree, and alkaline phosphatase activity. RNA/Co/chromatin immunoprecipitation and dual-luciferase assays and determination of mRNA stability and ubiquitination level showed that SNHG14 bound to human antigen R improves NEDD4L mRNA stability and expression, further promoted FOXA2 ubiquitination to inhibit FOXA2 expression, and then reduced FOXA2 enrichment on the PCP4 promoter to upregulate PCP4 transcription. Functional rescue experiments showed that the overexpression of NEDD4L or PCP4 and knockdown of FOXA2 both attenuated the inhibition of SNHG14 downregulation on OD of BMSCs. Overall, our findings suggested that SNHG14 promoted OD of BMSCs through the NEDD4L/FOXA2/PCP4 axis.

Genome-wide screening for genetic variants in polyadenylation signal (PAS) sites in mouse selection lines for fatness and leanness

Alternative polyadenylation (APA) determines mRNA stability, localisation, translation and protein function. Several diseases, including obesity, have been linked to APA. Studies have shown that single nucleotide polymorphisms in polyadenylation signals (PAS-SNPs) can influence APA and affect phenotype and disease susceptibility. However, these studies focussed on associations between single PAS-SNP alleles with very large effects and phenotype. Therefore, we performed a genome-wide screening for PAS-SNPs in the polygenic mouse selection lines for fatness and leanness by whole-genome sequencing. The genetic variants identified in the two lines were overlapped with locations of PAS sites obtained from the PolyASite 2.0 database. Expression data for selected genes were extracted from the microarray expression experiment performed on multiple tissue samples. In total, 682 PAS-SNPs were identified within 583 genes involved in various biological processes, including transport, protein modifications and degradation, cell adhesion and immune response. Moreover, 63 of the 583 orthologous genes in human have been previously associated with human diseases, such as nervous system and physical disorders, and immune, endocrine, and metabolic diseases. In both lines, PAS-SNPs have also been identified in genes broadly involved in APA, such as Polr2c, Eif3e and Ints11. Five PAS-SNPs within 5 genes (Car, Col4a1, Itga7, Lat, Nmnat1) were prioritised as potential functional variants and could contribute to the phenotypic disparity between the two selection lines. The developed PAS-SNPs catalogue presents a key resource for planning functional studies to uncover the role of PAS-SNPs in APA, disease susceptibility and fat deposition.

Ribosome slowdown triggers codon-mediated mRNA decay independently of ribosome quality control.

The control of mRNA stability plays a central role in regulating gene expression patterns. Recent studies have revealed that codon composition in the open reading frame determines mRNA stability in multiple organisms. Based on genome-wide correlation approaches, this previously unrecognized role for the genetic code is attributable to the kinetics of the codon-decoding process by the ribosome. However, complementary experimental analyses are required to clarify the codon effects on mRNA stability and the related cotranslational mRNA decay pathways, for example, those triggered by aberrant ribosome stalling. In the current study, we performed a set of reporter-based analyses to define codon-mediated mRNA decay and ribosome stall-dependent mRNA decay in zebrafish embryos. Our analysis showed that the effect of codons on mRNA stability stems from the decoding process, independent of the ribosome quality control factor Znf598 and stalling-dependent mRNA decay. We propose that codon-mediated mRNA decay is rather triggered by transiently slowed ribosomes engaging in a productive translation cycle in zebrafish embryos.

Pan-cancer analyses of synonymous mutations based on tissue-specific codon optimality

Codon optimality has been demonstrated to be an important determinant of mRNA stability and expression levels in multiple model organisms and human cell lines. However, tissue-specific codon optimality has not been developed to investigate how codon optimality is usually perturbed by somatic synonymous mutations in human cancers. Here, we determined tissue-specific codon optimality in 29 human tissues based on mRNA expression data from the Genotype-Tissue Expression project. We found that optimal codons were associated with differentiation, whereas non-optimal codons were correlated with proliferation. Furthermore, codons biased toward differentiation displayed greater tissue specificity in codon optimality, and the tissue specificity of codon optimality was primarily present in amino acids with high degeneracy of the genetic code. By applying tissue-specific codon optimality to somatic synonymous mutations in 8532 tumor samples across 24 cancer types and to those in 416 normal cells across six human tissues, we found that synonymous mutations frequently increased optimal codons in tumor cells and cancer-related genes (e.g., genes involved in cell cycle). Furthermore, an elevated frequency of optimal codon gain was found to promote tumor cell proliferation in three cancer types characterized by DNA damage repair deficiency and could act as a prognostic biomarker for patients with triple-negative breast cancer. In summary, this study profiled tissue-specific codon optimality in human tissues, revealed alterations in codon optimality caused by synonymous mutations in human cancers, and highlighted the non-negligible role of optimal codon gain in tumorigenesis and therapeutics.

Emerging roles of alternative cleavage and polyadenylation (APA) in human disease.

In the messenger RNA (mRNA) maturation process, the 3'-end of pre-mRNA is cleaved and a poly(A) sequence is added, this is an important determinant of mRNA stability and its cellular functions. More than 60%-70% of human genes have three or more polyadenylation (APA) sites and can be cleaved at different sites, generating mRNA transcripts of varying lengths. This phenomenon is termed as alternative cleavage and polyadenylation (APA) and it plays role in key biological processes like gene regulation, cell proliferation, senescence, and also in various human diseases. Loss of regulatory microRNA binding sites and interactions with RNA-binding proteins leading to APA are largely investigated in human diseases. However, the functions of the core APA machinery and related factors during disease conditions remain largely unknown. In this review, we discuss the roles of polyadenylation machinery in relation to brain disease, cardiac failure, pulmonary fibrosis, cancer, infectious conditions, and other human diseases. Collectively, we believe this review will be a useful avenue for understanding the emerging role of APA in the pathobiology of various human diseases.

Distinct Hypoxia-Induced Translational Profiles of Embryonic and Adult-Derived Macrophages

Tissue homeostasis and repair are orchestrated by resident and newly recruited macrophages that alter their gene expression program in response to changes in tissue microenvironment. Embryonic macrophages, such as fetal liver derived macrophages (FLDM) seed the organs, including heart and lung during embryonic development and persist throughout the adult lifetime, while bone marrow-derived macrophages (BMDM) are recruited following an acute perturbation. Transcriptome analyses of FLDM and BMDM identified differences between them at the level of RNA expression, which correlates imperfectly with protein levels. Post-transcriptional regulation by microRNAs (miRNAs) and RNA-binding proteins determines mRNA stability and translation rate and may override transcriptional cues in response to environmental changes, such as hypoxia. To identify distinct features of FLDM and BMDM response to hypoxia at the level of translation, we employed translating ribosome affinity purification (TRAP) to isolate polysomal RNA. RNA-seq profiling of translated RNA identified distinct hypoxia-induced translational signature of BMDM (Ly6e, vimentin and glycolysis-associated enzymes Pgk1, Tpi1, Aldoa, Ldha) and FLDM (chemokines Ccl7 and Ccl2). By translational profiling of BMDM and FLDM with deletion of the RNA-binding protein HuR, we identified transcripts that were dependent on HuR. These findings highlight the importance of HuR and identify its distinct targets for post-transcriptional regulation of gene expression in embryonic vs. adult-derived macrophages.

Crosstalk between codon optimality and cis-regulatory elements dictates mRNA stability

BackgroundThe regulation of messenger RNA (mRNA) stability has a profound impact on gene expression dynamics during embryogenesis. For example, in animals, maternally deposited mRNAs are degraded after fertilization to enable new developmental trajectories. Regulatory sequences in 3′ untranslated regions (3′UTRs) have long been considered the central determinants of mRNA stability. However, recent work indicates that the coding sequence also possesses regulatory information. Specifically, translation in cis impacts mRNA stability in a codon-dependent manner. However, the strength of this mechanism during embryogenesis, as well as its relationship with other known regulatory elements, such as microRNA, remains unclear.ResultsHere, we show that codon composition is a major predictor of mRNA stability in the early embryo. We show that this mechanism works in combination with other cis-regulatory elements to dictate mRNA stability in zebrafish and Xenopus embryos as well as in mouse and human cells. Furthermore, we show that microRNA targeting efficacy can be affected by substantial enrichment of optimal (stabilizing) or non-optimal (destabilizing) codons. Lastly, we find that one microRNA, miR-430, antagonizes the stabilizing effect of optimal codons during early embryogenesis in zebrafish.ConclusionsBy integrating the contributions of different regulatory mechanisms, our work provides a framework for understanding how combinatorial control of mRNA stability shapes the gene expression landscape.

Mechanism of Virus Attenuation by Codon Pair Deoptimization

Codon pair deoptimization is an efficient virus attenuation strategy, but the mechanism that leads to attenuation is unknown. The strategy involves synthetic recoding of viral genomes that alters the positions of synonymous codons, thereby increasing the number of suboptimal codon pairs and CpG dinucleotides in recoded genomes. Here we identify the molecular mechanism of codon pair deoptimization-based attenuation by studying recoded influenza A viruses. We show that suboptimal codon pairs cause attenuation, whereas the increase of CpG dinucleotides has no effect. Furthermore, we show that suboptimal codon pairs reduce both mRNA stability and translation efficiency of codon pair-deoptimized genes. Consequently, reduced protein production directly causes virus attenuation. Our study provides evidence that suboptimal codon pairs are major determinants of mRNA stability. Additionally, it demonstrates that codon pair bias canbe used to increase mRNA stability and protein production of synthetic genes in many areas of biotechnology.

Differences in Stability of Viral and Viral-Cellular Fusion Transcripts in HPV-Induced Cervical Cancers.

HPV-DNA integration results in dysregulation of viral oncogene expression. Because viral-cellular fusion transcripts inherently lack the viral AU-rich elements of the 3’UTR, they are considered to be more stable than episome-derived transcripts. The aim of this study is to provide formal proof for this assumption by comparing the stability of viral early transcripts derived from episomal and integrated HPV16 DNA, respectively. Full-length cDNA of three fusion transcripts comprising viral and cellular sequences in sense orientation were amplified and cloned into the adeno-viral-vector pAd/CMV/V5-DEST. The most abundant HPV16 oncogene transcript E6*I-E7-E1vE4-E5 with and without 3’UTR, served as reference and control, respectively. Human primary keratinocytes were transduced using high titer virus stocks. qRT-PCR was performed to determine mRNA stability in relation to GAPDH in the presence of actinomycin-D. In four independent transduction experiments, all three viral-cellular fusion transcripts were significantly more stable compared to the episome-derived reference. Among the three viral-cellular fusion transcripts the most stable transcript was devoid of the instability core motif “AUUUA”. Unexpectedly, there was no significant difference in the stability between the episome-derived transcripts either with or without 3’UTR, indicating that the AU-rich elements of the 3’UTR are not contributing to RNA stability. Instead, the three “AUUUA” motifs located in the untranslated region between the viral E4 and E5 genes may be responsible for the instability. This is the first report showing that authentic viral-cellular fusion transcripts are more stable than episome-derived transcripts. The longer half-life of the fusion transcripts may result in increased levels of viral oncoproteins and thereby drive the carcinogenic process.

Determining mRNA Stability by Metabolic RNA Labeling and Chemical Nucleoside Conversion.

The varying rates at which mRNAs decay are tightly coordinated with transcriptional changes to shape gene expression during development and disease. But currently available RNA sequencing approaches lack the temporal information to determine the relative contribution of RNA biogenesis, processing and turnover to the establishment of steady-state gene expression profiles.Here, we describe a protocol that combines metabolic RNA labeling with chemical nucleoside conversion by thiol-linked alkylation of 4-thiouridine to determine RNA stability in cultured cells (SLAMseq). When coupled to cost-effective mRNA 3' end sequencing approaches, SLAMseq determines the half-life of polyadenylated transcripts in a global and transcript-specific manner using untargeted or targeted cDNA library preparation protocols.We provide a step-by-step instruction for time-resolved mRNA 3' end sequencing, which augments traditional RNA-seq approaches to acquire the temporal resolution necessary to study the molecular principles that control gene expression.

Genome wide analysis of 3' UTR sequence elements and proteins regulating mRNA stability during maternal-to-zygotic transition in zebrafish.

Posttranscriptional regulation plays a crucial role in shaping gene expression. During the maternal-to-zygotic transition (MZT), thousands of maternal transcripts are regulated. However, how different cis-elements and trans-factors are integrated to determine mRNA stability remains poorly understood. Here, we show that most transcripts are under combinatorial regulation by multiple decay pathways during zebrafish MZT. By using a massively parallel reporter assay, we identified cis-regulatory sequences in the 3′ UTR, including U-rich motifs that are associated with increased mRNA stability. In contrast, miR-430 target sequences, UAUUUAUU AU-rich elements (ARE), CCUC, and CUGC elements emerged as destabilizing motifs, with miR-430 and AREs causing mRNA deadenylation upon genome activation. We identified trans-factors by profiling RNA–protein interactions and found that poly(U)-binding proteins are preferentially associated with 3′ UTR sequences and stabilizing motifs. We show that this activity is antagonized by C-rich motifs and correlated with protein binding. Finally, we integrated these regulatory motifs into a machine learning model that predicts reporter mRNA stability in vivo.

RNA-binding proteins distinguish between similar sequence motifs to promote targeted deadenylation by Ccr4-Not.

The Ccr4-Not complex removes mRNA poly(A) tails to regulate eukaryotic mRNA stability and translation. RNA-binding proteins contribute to specificity by interacting with both Ccr4-Not and target mRNAs, but this is not fully understood. Here, we reconstitute accelerated and selective deadenylation of RNAs containing AU-rich elements (AREs) and Pumilio-response elements (PREs). We find that the fission yeast homologues of Tristetraprolin/TTP and Pumilio/Puf (Zfs1 and Puf3) interact with Ccr4-Not via multiple regions within low-complexity sequences, suggestive of a multipartite interface that extends beyond previously defined interactions. Using a two-color assay to simultaneously monitor poly(A) tail removal from different RNAs, we demonstrate that Puf3 can distinguish between RNAs of very similar sequence. Analysis of binding kinetics reveals that this is primarily due to differences in dissociation rate constants. Consequently, motif quality is a major determinant of mRNA stability for Puf3 targets in vivo and can be used for the prediction of mRNA targets.

Non-invasive measurement of mRNA decay reveals translation initiation as the major determinant of mRNA stability.

The cytoplasmic abundance of mRNAs is strictly controlled through a balance of production and degradation. Whereas the control of mRNA synthesis through transcription has been well characterized, less is known about the regulation of mRNA turnover, and a consensus model explaining the wide variations in mRNA decay rates remains elusive. Here, we combine non-invasive transcriptome-wide mRNA production and stability measurements with selective and acute perturbations to demonstrate that mRNA degradation is tightly coupled to the regulation of translation, and that a competition between translation initiation and mRNA decay -but not codon optimality or elongation- is the major determinant of mRNA stability in yeast. Our refined measurements also reveal a remarkably dynamic transcriptome with an average mRNA half-life of only 4.8 min - much shorter than previously thought. Furthermore, global mRNA destabilization by inhibition of translation initiation induces a dose-dependent formation of processing bodies in which mRNAs can decay over time.

Modulating Heterologous Gene Expression with Portable mRNA-Stabilizing 5'-UTR Sequences.

RNA half-lives are frequently perceived as depending on too many variables, and transcript stability is generally missed as a checkpoint amenable to manipulation in synthetic designs. In this work, the contribution of mRNA stability to heterologous protein production levels in E.coli has been inspected. To this end, we capitalized on the wealth of information available on intrinsic mRNA stability determinants, four of which were formatted as portable modules consisting of 5'-untranslated regions (UTRs). The cognate DNA sequences were then assembled in a genetic frame in which mRNA stability endowed by the UTRs was the only variable to run expression of sfGFP. Reporter output and Northern blot-based measurements of absolute mRNA half-lives revealed that such UTRs were found to keep intact their ability to modulate transcript stability when excised from their natural context and placed as the upstream region of the reporter gene. By keeping transcription fixed and combining different UTRs with a constant ribosomal binding site, we showed that mRNA decay can be made the limiting constituent of the overall gene expression flow. Moreover, the data indicated that manipulating mRNA stability had little effect on expression noise in the corresponding population. Finally, augmented heterologous expression brought about by mRNA stability did not make cells more vulnerable to resource-consuming stresses. The tangible result of this work was a collection of well-characterized mRNA-stabilizing sequences that can be composed along with other expression signals in any construct following the assembly rules of the Standard European Vector Architecture (SEVA) format.

Codon Usage and Amino Acid Identity Are Major Determinants of MRNA Stability in Humans

Codon Usage and Amino Acid Identity Are Major Determinants of MRNA Stability in Humans

MRNA Stability Assay Using transcription inhibition by Actinomycin D in Mouse Pluripotent Stem Cells.

Gene expression is regulated through multiple steps at both transcriptional and post-transcriptional levels. The net abundance of mature mRNA species in cells is determined by the balance between transcription and degradation. Thus, the regulation of mRNA stability is a key post-transcriptional event that can greatly affect the net level of mRNAs in cells. The mRNA stability within cells can be measured indirectly by analyzing the mRNA half-life following transcription inhibition, where changes in mRNA levels are assumed to reflect mRNA degradation. Determination of mRNA half-life as a measure of mRNA stability is useful in understanding gene expression changes and underlying mechanisms regulating the level of transcripts at different physiological conditions or developmental stages. The protocol described here presents the analysis of mRNA decay as a measure for determining mRNA stability after transcriptional inhibition with Actinomycin D treatment in control and SRSF3 depleted mouse induced pluripotent stem cells (iPSC).

Impact of Methods on the Measurement of mRNA Turnover.

The turnover of the RNA molecules is determined by the rates of transcription and RNA degradation. Several methods have been developed to study RNA turnover since the beginnings of molecular biology. Here we summarize the main methods to measure RNA half-life: transcription inhibition, gene control, and metabolic labelling. These methods were used to detect the cellular activity of the mRNAs degradation machinery, including the exo-ribonuclease Xrn1 and the exosome. On the other hand, the study of the differential stability of mature RNAs has been hampered by the fact that different methods have often yielded inconsistent results. Recent advances in the systematic comparison of different method variants in yeast have permitted the identification of the least invasive methodologies that reflect half-lives the most faithfully, which is expected to open the way for a consistent quantitative analysis of the determinants of mRNA stability.

Determining mRNA half-lives on a transcriptome-wide scale.

Every step in the life cycle of an RNA transcript provides opportunity for regulation. One important aspect of post-transcriptional control is the regulation of RNA stability. Of the many strategies for determining mRNA stability, transcription inhibition and metabolic labeling have proved the most amenable to high-throughput analysis and have opened the door to dissecting mRNA decay transcriptome-wide. Here, we describe experimental and computational methods to determine transcriptome-wide RNA stabilities using both pharmacological inhibition of transcription and metabolic labeling. To aid in the analysis of these experiments, we discuss key characteristics of high-quality experiments and address other experimental and computational considerations for the analysis of mRNA stability. Broader application of these approaches will further our understanding of mRNA decay and illuminate its contribution to different biological processes.

Cis-regulatory elements explain most of the mRNA stability variation across genes in yeast.

The stability of mRNA is one of the major determinants of gene expression. Although a wealth of sequence elements regulating mRNA stability has been described, their quantitative contributions to half-life are unknown. Here, we built a quantitative model for Saccharomyces cerevisiae based on functional mRNA sequence features that explains 59% of the half-life variation between genes and predicts half-life at a median relative error of 30%. The model revealed a new destabilizing 3′ UTR motif, ATATTC, which we functionally validated. Codon usage proves to be the major determinant of mRNA stability. Nonetheless, single-nucleotide variations have the largest effect when occurring on 3′ UTR motifs or upstream AUGs. Analyzing mRNA half-life data of 34 knockout strains showed that the effect of codon usage not only requires functional decapping and deadenylation, but also the 5′-to-3′ exonuclease Xrn1, the nonsense-mediated decay genes, but not no-go decay. Altogether, this study quantitatively delineates the contributions of mRNA sequence features on stability in yeast, reveals their functional dependencies on degradation pathways, and allows accurate prediction of half-life from mRNA sequence.