RNA Regulatory Elements Research Articles

MiR-130a/TGF-β1 axis is involved in sow fertility by controlling granulosa cell apoptosis

TGF-β1 is a ligand of the TGF-β superfamily and an important cytokine that regulates ovarian functions including follicular development, steroid production, ovulation, luteinization, and female fertility. However, little is known about the regulation of TGF-β1 expression in ovary. Here, we identified that TGF-β1 is a functional target of miR-130a in porcine ovarian granulosa cells (GCs). The 3′-UTR sequence of TGF-β1 gene (1137 bp in length) in Large White (LW) pig was isolated, and multiple RNA regulatory elements (RREs), including several binding motifs of different miRNAs, were identified in this region. Luciferase activity assay showed that miR-130a dramatically suppresses the 3′-UTR luciferase activity of TGF-β1 gene, and further inhibits the expression of TGF-β1 in porcine GCs. FACS revealed that miR-130a acts as a pro-apoptotic factor and promotes GC apoptosis by inhibiting TGF-β1. Two novel linked mutations (−573G > A and −540T > C) were identified in the promoter region of ssc-miR-130a, but their polymorphisms are not associated with sow reproductive traits. Importantly, combined genotype analysis with a known mutation (c.1583 A > G) in the 3′-UTR of porcine TGF-β1 gene showed a significant association with reproductive performance in LW sow population. Overall, our findings defined a novel regulatory axis, miR-130a/TGF-β1 axis, which is involved in regulating sow fertility.

Abstract 6326: Global changes in alternative mRNA polyadenylation profile promote the development of multidrug resistance (MDR) to chemotherapy in colon cancer

Abstract Aim: The study investigated the global changes in mRNA alternative polyadenylation (APA) profile associated with MDR development in colon cancer cells. Background: Polyadenylation is a crucial step needed for the maturation of eukaryotic mRNAs. APA refers to the production of different mRNA isoforms bearing varying length of 3'-untranslated region (3'UTR), where key RNA regulatory elements are found. APA usually eliminates the distal end of the 3'UTR, thus allowing the escape from the predominantly repressive effect from longer 3'UTR. Colon cancer is a leading cause of cancer-related death worldwide. MDR, which is usually associated with overexpression of ATP-binding cassette efflux transporters including ABCG2, is a major unresolved obstacle to successful cancer chemotherapy. We have previously reported that ABCG2 mRNA is more stable in MDR colon cancer cells. Interestingly, the increase in mRNA stability is tied to a missing microRNA binding site (miR-519c) due to the shortening of ABCG2 mRNA 3'UTR in drug resistant cells. Thus microRNA-mediated mRNA degradation and/or protein translation block are relieved, thereby leading to ABCG2 overexpression. A remarkable feature of 3'UTR APA is that it can be regulated globally, thus simultaneously affecting numerous mRNA transcripts. While ABCG2 mRNA 3'UTR is shortened in MDR colon cancer cells, the global changes in APA profile of other transcripts during the course of MDR development have not been reported. Method: Differential APA signature and specific APA-regulated mRNAs were evaluated in two pairs of sensitive and MDR colon cancer cell lines by RNA sequencing. APA profiles were assessed from the RNA-Seq datasets by using the bioinformatics tool “DaPars”. Altered 3'UTR isoforms of a few differentially expressed mRNAs were validated by 3'RACE assay and 3'UTR isoform specific PCR analysis. The genes were subjected to Gene Ontology (GO) analysis to identify relevant molecular pathways and biological processes. Result: APA profiles of two pairs of drug resistant ABCG2-overexpressing colon cancer cell line (S1-M1-80 & SW620 Flv100) versus their parental cells (S1 & SW620) were compared at the global scale from their sequenced RNA samples. By GO analysis using the PANTHER Platform, mRNA transcripts exhibiting 3'UTR shortening in S1-M1-80 and SW620 Flv100 versus their respective parental cells were enriched in biological processes including “transport and catabolism”, “cell growth and death”, and “cell motility” by GO terms. Genes ranked at the top of the list are mostly direct or indirect targets of the Wnt-β-catenin signaling pathway. Wnt signaling is a crucial pathway for mediating metastasis and therapeutic resistance. Conclusion: Global changes of APA in gene regulation may contribute to the development of MDR in colon cancer. Further investigation is warranted to elucidate the mechanisms leading to APA. Modulation of the APA machinery may help eradicate MDR cancer cells. Citation Format: Kenneth KW To. Global changes in alternative mRNA polyadenylation profile promote the development of multidrug resistance (MDR) to chemotherapy in colon cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6326.

Investigation of Novel RNA Elements in the 3'UTR of Tobacco Necrosis Virus-D.

RNA elements in the untranslated regions of plus-strand RNA viruses can control a variety of viral processes including translation, replication, packaging, and subgenomic mRNA production. The 3′ untranslated region (3′UTR) of Tobacco necrosis virus strain D (TNV-D; genus Betanecrovirus, family Tombusviridae) contains several well studied regulatory RNA elements. Here, we explore a previously unexamined region of the viral 3′UTR, the sequence located upstream of the 3′-cap independent translation enhancer (3′CITE). Our results indicate that (i) a long-range RNA–RNA interaction between an internal RNA element and the 3′UTR facilitates translational readthrough, and may also promote viral RNA synthesis; (ii) a conserved RNA hairpin, SLX, is required for efficient genome accumulation; and (iii) an adenine-rich region upstream of the 3′CITE is dispensable, but can modulate genome accumulation. These findings identified novel regulatory RNA elements in the 3′UTR of the TNV-D genome that are important for virus survival.

Expression of ribosomal protection protein RppA is regulated by a ribosome-dependent ribo-regulator and two mistranslation products.

Gene expression is tightly controlled by transcription factors and RNA regulatory elements, including trans-acting small RNAs, cis-regulatory riboswitches and ribosome-dependent ribo-regulators. In the present study, we demonstrated that a ribosome-dependent ribo-regulator and two mistranslation products co-regulate rppA (encoding a ribosomal protection protein) expression in Bacillus thuringiensis BMB171. The leader RNA of the rppA gene controls rppA expression via translation of leader ORF1 resident in its sequence. In the presence of chloramphenicol, a +1 frameshift product (ORF2) and a stop codon readthrough product (ORF3) of ORF1 emerged. ORF3 exerted a negative effect on rppA expression. By contrast, the ORF2 promoted rppA expression. The regulation mode identified in the present study will lead to a deeper understanding of bacterial gene expression.

Development of Novel Riboswitches for Synthetic Biology in the Green Alga Chlamydomonas.

Riboswitches are RNA regulatory elements that bind specific ligands to control gene expression. Because of their modular composition, where a ligand-sensing aptamer domain is combined with an expression platform, riboswitches offer unique tools for synthetic biology applications. Here we took a mutational approach to determine functionally important nucleotide residues in the thiamine pyrophosphate (TPP) riboswitch in the THI4 gene of the model alga Chlamydomonas reinhardtii, allowing us to carry out aptamer swap using THIC aptamers from Chlamydomonas and Arabidopsis thaliana. These chimeric riboswitches displayed a distinct specificity and dynamic range of responses to different ligands. Our studies demonstrate ease of assembly as 5′UTR DNA parts, predictability of output, and utility for controlled production of a high-value compound in Chlamydomonas. The simplicity of riboswitch incorporation in current design platforms will facilitate the generation of genetic circuits to advance synthetic biology and metabolic engineering of microalgae.

An mRNA-mRNA Interaction Couples Expression of a Virulence Factor and Its Chaperone in Listeria monocytogenes

SUMMARYBacterial pathogens often employ RNA regulatory elements located in the 5′ untranslated regions (UTRs) to control gene expression. Using a comparative structural analysis, we examine the structure of 5′ UTRs at a global scale in the pathogenic bacterium Listeria monocytogenes under different conditions. In addition to discovering an RNA thermoswitch and detecting simultaneous interaction of ribosomes and small RNAs with mRNA, we identify structural changes in the 5′ UTR of an mRNA encoding the post-translocation chaperone PrsA2 during infection conditions. We demonstrate that the 5′ UTR of the prsA2 mRNA base pairs with the 3′ UTR of the full-length hly mRNA encoding listeriolysin O, thus preventing RNase J1-mediated degradation of the prsA2 transcript. Mutants lacking the hly-prsA2 interaction exhibit reduced virulence properties. This work highlights an additional level of RNA regulation, where the mRNA encoding a chaperone is stabilized by the mRNA encoding its substrate.

Characterization of a transcriptional TPP riboswitch in the human pathogen Neisseria meningitidis

ABSTRACTIncreasing evidence has demonstrated that regulatory RNA elements such as riboswitches (RS) play a pivotal role in the fine-tuning of bacterial gene expression. In this study, we investigated and characterized a novel transcriptional thiamine pyrophosphate (TPP) RS in the obligate human pathogen N. meningitidis MC58 (serogroup B). This RS is located in the 5´ untranslated region upstream of thiC gene, encoding a protein involved in TPP biosynthesis, an essential cofactor for all living beings. Primer extension revealed the transcriptional start site of thiC. Northern blot analysis of thiC mRNA and reporter gene studies confirmed the presence of an active TPP-sensing RS. Expression patterns of the wild-type RS and site-specific mutants showed that it is an OFF switch that controls transcription elongation of thiC mRNA. Interestingly, the regulatory mechanism of the meningococcal thiC RS resembles the Gram-positive Bacillus subtilis thiC RS rather than the Gram-negative Escherichia coli thiC RS. Therefore, the meningococcal thiC RS represents a rare example of transcriptional RS in a Gram-negative bacterium. We further observed that the RS is actively involved in modulating gene expression in response to different growth media and to supplemented bacterial and eukaryotic cell lysates as possible sources of nutrients in the nasopharynx. Our results suggest that RS-mediated gene regulation could influence meningococcal fitness, through the fine-tuning of biosynthesis and scavenging of nutrients and cofactors, such as thiamine.

The cell stress response: extreme times call for post-transcriptional measures.

Following cell stress, a wide range of molecular pathways are initiated to orchestrate the stress response and enable adaptation to an environmental or intracellular perturbation. The post-transcriptional regulation strategies adopted during the stress response result in a substantial reorganization of gene expression, designed to prepare the cell for either acclimatization or programmed death, depending on the nature and intensity of the stress. Fundamental to the stress response is a rapid repression of global protein synthesis, commonly mediated by phosphorylation of translation initiation factor eIF2α. Recent structural and biochemical information have added unprecedented detail to our understanding of the molecular mechanisms underlying this regulation. During protein synthesis inhibition, the translation of stress-specific mRNAs is nonetheless enhanced, often through the interaction between RNA-binding proteins and specific RNA regulatory elements. Recent studies investigating the unfolded protein response (UPR) provide some important insights into how posttranscriptional events are spatially and temporally fine-tuned in order to elicit the most appropriate response and to coordinate the transition from an early, acute stage into the chronic state of adaptation. Importantly, cancer cells are known to hi-jack adaptive stress response pathways, particularly the UPR, to survive and proliferate in the unfavorable tumor environment. In this review, we consider the implications of recent research into stress-dependent post-transcriptional regulation and make the case for the exploration of the stress response as a strategy to identify novel targets in the development of cancer therapies. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution Translation > Translation Mechanisms > Translation Regulation.

Variants in BMP7 and BMP15 3'-UTRs Associated with Reproductive Traits in a Large White Pig Population.

Bone morphogenetic protein 7 (BMP7) and BMP15, which encode members of the BMP family, have been identified by whole-genome resequencing as breeding-related genes that overlap with a known quantitative trait locus for reproductive traits. In this study, we investigated the effects of variants at the BMP7 and BMP15 gene loci on sow reproductive traits. We isolated 669 and 1213 bp sequences of the 3’-untranslated region (3’-UTR) of the porcine BMP7 and BMP15 genes, respectively, and detected several RNA regulatory elements, such as miRNA response elements and AU-rich elements. Pooled DNA sequencing identified two novel point mutations (viz., BMP7 c.1569A>G and BMP15 c.2366G>A) in the 3’-UTR. Association analysis showed that the c.1569A>G polymorphism was associated with the litter weight trait in a Large White pig population. Furthermore, analysis of the combined genetic effects revealed that AA/GA and AG/GG were the favorable combined genotypes for the total number of piglets born (TNB) and the total number of piglets born alive (NBA), whereas. Together, our findings confirm that BMP7 and BMP15 are candidate genes for porcine reproductive performance.

The Hepatitis E Virus Rna Regulatory Elements

The Hepatitis E Virus Rna Regulatory Elements

Self-powered RNA nanomachine driven by metastable structure.

Many non-coding and regulatory RNA elements have evolved to exploit transient or metastable structures that emerge during transcription to control complex folding pathways or to encode dynamic functions. However, efforts to engineer synthetic RNA devices have mostly focused on the thermodynamically stable structures. Consequently, significant challenges and opportunities exist in engineering functional RNAs that explicitly take advantage of cotranscriptionally generated transient or metastable structures. In this work, we designed a short RNA sequence that adopts a robust metastable structure when transcribed by an RNA polymerase. Although the metastable structure persists for hours at low temperature, it refolds almost completely into the thermodynamically stable structure upon heat denaturation followed by cooling. The synthetic RNA was also equipped with the Broccoli aptamer so that it can bind its ligand and become fluorescent only in the thermodynamically stable structure. We further demonstrated that the relaxation to the thermodynamically stable and fluorescent structure can be catalyzed by a short trigger RNA in a sequence-specific manner. Finally, the RNA architecture was redesigned to sense and respond to microRNA sequences. In summary, we designed RNA nanomachines that can detect an RNA sequence, amplify signal and produce an optical output, all encoded in a single RNA transcript, self-powered by a metastable structure.

DrosophilaSister-of-Sex-lethal reinforces a male-specific gene expression pattern by controllingSex-lethalalternative splicing

In Drosophila, female development is governed by a single RNA-binding protein, Sex-lethal (Sxl), that controls the expression of key factors involved in dosage compensation, germline homeostasis and the establishment of female morphology and behaviour. Sxl expression in female flies is maintained by an auto-regulatory, positive feedback loop with Sxl controlling splicing of its own mRNA. Until now, it remained unclear how males prevent accidental triggering of the Sxl expression cascade and protect themselves against runaway protein production. Here, we identify the protein Sister-of-Sex-lethal (Ssx) as an inhibitor of Sxl auto-regulatory splicing. Sxl and Ssx have a comparable RNA-binding specificity and compete for binding to RNA regulatory elements present in the Sxl transcript. In cultured Drosophila cells, Sxl-induced changes to alternative splicing can be reverted by the expression of Ssx. Moreover, in adult male flies ablation of the ssx gene results in a low level of productive Sxl mRNA splicing and Sxl protein production in isolated, clonal cell populations. In sum, this demonstrates that Ssx safeguards male animals against Sxl protein production to reinforce a stable, male-specific gene expression pattern.

RNA Structure Elements Conserved between Mouse and 59 Other Vertebrates.

In this work, we present a computational screen conducted for functional RNA structures, resulting in over 100,000 conserved RNA structure elements found in alignments of mouse (mm10) against 59 other vertebrates. We explicitly included masked repeat regions to explore the potential of transposable elements and low-complexity regions to give rise to regulatory RNA elements. In our analysis pipeline, we implemented a four-step procedure: (i) we screened genome-wide alignments for potential structure elements using RNAz-2, (ii) realigned and refined candidate loci with LocARNA-P, (iii) scored candidates again with RNAz-2 in structure alignment mode, and (iv) searched for additional homologous loci in mouse genome that were not covered by genome alignments. The 3’-untranslated regions (3’-UTRs) of protein-coding genes and small noncoding RNAs are enriched for structures, while coding sequences are depleted. Repeat-associated loci make up about 95% of the homologous loci identified and are, as expected, predominantly found in intronic and intergenic regions. Nevertheless, we report the structure elements enriched in specific genome elements, such as 3’-UTRs and long noncoding RNAs (lncRNAs). We provide full access to our results via a custom UCSC genome browser trackhub freely available on our website (http://rna.tbi.univie.ac.at/trackhubs/#RNAz).

Polymorphism studies on microRNA targetome of thalassemia.

Thalassemia is one of the most prevalent hemoglobin disorders. It is caused by the decreased or absent synthesis of one globin chain that leads to moderate to severe hemolytic anemia in clinical complications. Some genetic factors cause these phenotypic variations by the alteration of gene expression. MicroRNAs (miRNAs) are post-transcriptional regulators in gene expression. Therefore, variations in 3'-untranslated region (3'-UTR) of target genes may affect gene expression. It is of interest to evaluate the impact of noncoding SNPs in thalassemia related genes on miRNA: mRNA interactions in the severity of thalassemia. Polymorphisms that alter miRNA: mRNA interactions were predicted using PolymiRTS and Mirsnpscore tools. Then, the effect of predicted target SNPs on thermodynamic stability, local RNA structure and regulatory elements was investigated using RNAhybrid, RNAsnp and RegulomeDB, respectively. The molecular functions and the Biological process of candidate genes were extracted and interaction network was created. Forty-six SNPs were predicted to affect 188 miRNA interactions. These results suggest that 3'-UTR SNP may affect gene expression and cause phenotypic variation in thalassemia patients.

Riboswitches and Translation Control.

A growing collection of bacterial riboswitch classes is being discovered that sense central metabolites, coenzymes, and signaling molecules. Included among the various mechanisms of gene regulation exploited by these RNA regulatory elements are several that modulate messenger RNA (mRNA) translation. In this review, the mechanisms of riboswitch-mediated translation control are summarized to highlight both their diversity and potential ancient origins. These mechanisms include ligand-gated presentation or occlusion of ribosome-binding sites, control of alternative splicing of mRNAs, and the regulation of mRNA stability. Moreover, speculation on the potential for novel riboswitch discoveries is presented, including a discussion on the potential for the discovery of a greater diversity of mechanisms for translation control.

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs.

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding-a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

Bacterial riboswitches and RNA thermometers: Nature and contributions to pathogenesis.

Bacterial pathogens are always challenged by fluctuations of chemical and physical parameters that pose serious threats to cellular integrity and metabolic status. Sudden deprivation of nutrients or key metabolites, changes in surrounding pH, and temperature shifts are the most important examples of such parameters. To elicit a proper response to such fluctuations, bacterial cells coordinate the expression of parameter-relevant genes. Although protein-mediated control of gene expression is well appreciated since many decades, RNA-based regulation has been discovered in early 2000s as a parallel level of regulation. Small regulatory RNAs have emerged as one of the most widespread and important gene regulatory systems in bacteria with rare representatives found in Archaea and Eukarya. Riboswitches and thermosensors are cis-encoded RNA regulatory elements that employ different mechanisms to regulate the expression of related genes controlling key metabolic pathways and genes of temperature relevant proteins including virulence factors. The extent of RNA contributions to gene regulation is not completely known even in well-studied models such E. coli and B. subtilis. In depth understanding of riboswitches is promising for opportunity to discover a narrow spectrum antibacterial drugs that target riboswitches of essential metabolic pathways.

New tRNA contacts facilitate ligand binding in a Mycobacterium smegmatis T box riboswitch

T box riboswitches are RNA regulatory elements widely used by organisms in the phyla Firmicutes and Actinobacteria to regulate expression of amino acid-related genes. Expression of T box family genes is down-regulated by transcription attenuation or inhibition of translation initiation in response to increased charging of the cognate tRNA. Three direct contacts with tRNA have been described; however, one of these contacts is absent in a subclass of T box RNAs and the roles of several structural domains conserved in most T box RNAs are unknown. In this study, structural elements of a Mycobacterium smegmatis ileS T box riboswitch variant with an Ultrashort (US) Stem I were sequentially deleted, which resulted in a progressive decrease in binding affinity for the tRNAIle ligand. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) revealed structural changes in conserved riboswitch domains upon interaction with the tRNA ligand. Cross-linking and mutational analyses identified two interaction sites, one between the S-turn element in Stem II and the T arm of tRNAIle and the other between the Stem IIA/B pseudoknot and the D loop of tRNAIle These newly identified RNA contacts add information about tRNA recognition by the T box riboswitch and demonstrate a role for the S-turn and pseudoknot elements, which resemble structural elements that are common in many cellular RNAs.

In cell mutational interference mapping experiment (in cell MIME) identifies the 5' polyadenylation signal as a dual regulator of HIV-1 genomic RNA production and packaging.

Non-coding RNA regulatory elements are important for viral replication, making them promising targets for therapeutic intervention. However, regulatory RNA is challenging to detect and characterise using classical structure-function assays. Here, we present in cell Mutational Interference Mapping Experiment (in cell MIME) as a way to define RNA regulatory landscapes at single nucleotide resolution under native conditions. In cell MIME is based on (i) random mutation of an RNA target, (ii) expression of mutated RNA in cells, (iii) physical separation of RNA into functional and non-functional populations, and (iv) high-throughput sequencing to identify mutations affecting function. We used in cell MIME to define RNA elements within the 5′ region of the HIV-1 genomic RNA (gRNA) that are important for viral replication in cells. We identified three distinct RNA motifs controlling intracellular gRNA production, and two distinct motifs required for gRNA packaging into virions. Our analysis reveals the 73AAUAAA78 polyadenylation motif within the 5′ PolyA domain as a dual regulator of gRNA production and gRNA packaging, and demonstrates that a functional polyadenylation signal is required for viral packaging even though it negatively affects gRNA production.

HnRNP G prevents inclusion on the HPV16 L1 mRNAs of the central exon between splice sites SA3358 and SD3632.

HPV16 late L1 mRNAs encode a short central exon that is located between HPV16 3'-splice site SA3358 and HPV16 5'-splice site SD3632. While SA3358 is used to produce both HPV16 early mRNAs encoding the E6 and E7 oncogenes, and late mRNAs encoding E4, L1 and L2, SD3632 is used exclusively to produce late L1 mRNA. We have previously identified an 8-nucleotide regulatory RNA element that is required for inclusion of the exon between SA3358 and SD3632 to produce L1 mRNAs at the expense of mRNAs polyadenylated at the HPV16 early polyadenylation signal pAE. Here we show that this HPV16 8-nucleotide splicing enhancer interacts with hnRNP G. Binding of hnRNP G to this element prevents inclusion of the exon between SA3358 and SD3632 on the HPV16 late L1 mRNAs. We concluded that hnRNP G has a splicing inhibitory role and that hnRNP G can control HPV16 mRNA splicing.