UTR Of mRNA Research Articles

Insulin-like growth factor 2 mRNA-binding protein 2-regulated alternative splicing of nuclear factor 1 C-type causes excessive granulosa cell proliferation in polycystic ovary syndrome.

ObjectivesPolycystic ovary syndrome (PCOS) is a common reproductive endocrine disorder. Insulin‐like growth factor 2 mRNA‐binding protein 2 (IGF2BP2) serves as an HMGA2 target gene to promote the proliferation of granulosa cells (GCs). However, it is still unclear whether IGF2BP2 participates in the pathogenesis of PCOS as RNA binding protein (RBP). In this study, we aimed to elucidate IGF2BP2‐interacting transcripts, global transcriptome together with alternative splicing in GCs to eventually uncover potential mechanisms of PCOS pathogenesis.Materials and MethodsThe expression of IGF2BP2 in GCs from PCOS patients was detected using quantitative reverse transcription PCR (RT‐qPCR) and western blot. We captured IGF2BP2‐interacting transcripts, global transcriptome together with alternative splicing by RNA immunoprecipitation sequencing (RIP‐seq) and RNA sequencing (RNA‐seq). KGN cells transfected with IGF2BP2 overexpressing plasmids and nuclear factor 1 C‐type (NFIC) siRNAs, were applied to CCK‐8, EdU and TUNEL assays.ResultsIGF2BP2 was highly expressed in GCs from PCOS patients. As an RBP, it preferentially bound to the 3′and 5′UTRs of mRNAs with GGAC motif and a newly found GAAG motif. The overexpression of IGF2BP2 changed the transcriptome profile of KGN cells. IGF2BP2 functioned to regulate alternative splicing events and promote cell proliferation through inhibiting exon skipping events of NFIC.ConclusionIn conclusion, we demonstrated that IGF2BP2 promotes GC proliferation via regulating alternative splicing of NFIC in PCOS. The findings help to better understand the roles of IGF2BP2 in the pathogenesis of PCOS.

Rocaglates as Antivirals: Comparing the Effects on Viral Resistance, Anti-Coronaviral Activity, RNA-Clamping on eIF4A and Immune Cell Toxicity.

Rocaglates are potent broad-spectrum antiviral compounds with a promising safety profile. They inhibit viral protein synthesis for different RNA viruses by clamping the 5′-UTRs of mRNAs onto the surface of the RNA helicase eIF4A. Apart from the natural rocaglate silvestrol, synthetic rocaglates like zotatifin or CR-1-31-B have been developed. Here, we compared the effects of rocaglates on viral 5′-UTR-mediated reporter gene expression and binding to an eIF4A-polypurine complex. Furthermore, we analyzed the cytotoxicity of rocaglates on several human immune cells and compared their antiviral activities in coronavirus-infected cells. Finally, the potential for developing viral resistance was evaluated by passaging human coronavirus 229E (HCoV-229E) in the presence of increasing concentrations of rocaglates in MRC-5 cells. Importantly, no decrease in rocaglate-sensitivity was observed, suggesting that virus escape mutants are unlikely to emerge if the host factor eIF4A is targeted. In summary, all three rocaglates are promising antivirals with differences in cytotoxicity against human immune cells, RNA-clamping efficiency, and antiviral activity. In detail, zotatifin showed reduced RNA-clamping efficiency and antiviral activity compared to silvestrol and CR-1-31-B, but was less cytotoxic for immune cells. Our results underline the potential of rocaglates as broad-spectrum antivirals with no indications for the emergence of escape mutations in HCoV-229E.

Caveolin-1 peptide regulates p53-microRNA-34a feedback in fibrotic lung fibroblasts

SummaryIdiopathic pulmonary fibrosis (IPF) is a life-threatening disease resulting from dysregulated repair responses to lung injury. Excessive extracellular matrix deposition by expanding myofibroblasts and fibrotic lung fibroblasts (fLfs) has been implicated in the pathogenesis of PF, including IPF. We explored fLfs' microRNA-34a (miR-34a) expression from IPF tissues. Basal miR-34a levels were decreased with reduced binding of p53 to the promoter DNA and 3′UTR mRNA sequences. Overexpression of miR-34a in fLfs increased p53, PAI-1, and reduced pro-fibrogenic markers. The regulatory effects of miR-34a were altered by modifying the p53 expression. Precursor-miR-34a lung transduction reduced bleomycin-induced PF in wild-type mice. fLfs treated with caveolin-1 scaffolding domain peptide (CSP) or its fragment, CSP7, restored miR-34a, p53, and PAI-1. CSP/CSP7 reduced PDGFR-β and pro-fibrogenic markers, which was abolished in fLfs following blockade of miR-34a expression. These peptides failed to resolve PF in mice lacking miR-34a in fLfs, indicating miR-34a-p53-feedback induction required for anti-fibrotic effects.

Reversal of G-Quadruplexes' Role in Translation Control When Present in the Context of an IRES.

G-quadruplexes (GQs) are secondary nucleic acid structures that play regulatory roles in various cellular processes. G-quadruplex-forming sequences present within the 5′ UTR of mRNAs can function not only as repressors of translation but also as elements required for optimum function. Based upon previous reports, the majority of the 5′ UTR GQ structures inhibit translation, presumably by blocking the ribosome scanning process that is essential for detection of the initiation codon. However, there are certain mRNAs containing GQs that have been identified as positive regulators of translation, as they are needed for translation initiation. While most cellular mRNAs utilize the 5′ cap structure to undergo cap-dependent translation initiation, many rely on cap-independent translation under certain conditions in which the cap-dependent initiation mechanism is not viable or slowed down, for example, during development, under stress and in many diseases. Cap-independent translation mainly occurs via Internal Ribosomal Entry Sites (IRESs) that are located in the 5′ UTR of mRNAs and are equipped with structural features that can recruit the ribosome or other factors to initiate translation without the need for a 5′ cap. In this review, we will focus only on the role of RNA GQs present in the 5′ UTR of mRNAs, where they play a critical role in translation initiation, and discuss the potential mechanism of this phenomenon, which is yet to be fully delineated.

MiR-128-1-5p regulates differentiation of ovine stromal vascular fraction by targeting the KLF11 5′-UTR

Fat content is an important index to evaluate the individual performance of livestock animals such as sheep for meat production purposes. Reducing the subcutaneous and visceral fat while increasing the intramuscular fat is a valuable goal to achieve for the meat production industry. Here, we investigated the effect of miR-128-1-5p on adipogenesis of subcutaneous fat by targeting 5’-UTR in KLF11, a rare mechanism where most miRNAs bind the 3’-UTR of mRNAs. A dual fluorescence reporter assay was conducted to validate the binding sites of miR-128-1-5p on 5’-UTR of KLF11 mRNA. Roles of miR-128-1-5p in KLF11 expression were measured through co-transfecting miRNA mimics with KLF11-expressing vectors (CDSs together with or without the 5′-UTR) into ovine stromal vascular fractions (SVF). Additionally, functional roles of miR-128-1-5p, and KLF11 in adipogenesis of ovine subcutaneous fat were investigated. Results showed that miR-128-1-5p targeted KLF11 5′-UTR, reduced the fluorescence activity of the dual fluorescent reporter vector, as well as KLF11 mRNA, and protein expression levels. During the differentiation of SVF, disturbing the expression of miR-128-1-5p and KLF11 changed the adipogenic differentiation of SVF as observed in the lipid formation, and adipogenic marker genes. This study indicates that miR-128-1-5p promotes the expression of lipogenic marker genes and the formation of lipid droplets by targeting KLF11 5′-UTR. Furthermore, overexpression, and inhibition of KLF11 indicate that KLF11 inhibited SVF differentiation. In summary, the 5’-UTR binding mechanism discovered in this study extends the understanding of miRNA functions. Key roles of miR-128-1-5p and KLF11 in the adipogenesis of sheep subcutaneous fat have potential values for improving the meat and/or fat ratio of domestic animals.

Linking translation factor conformational dynamics with translation efficiencies and gene expression

Translation initiation is a tightly regulated step in eukaryotic translation that involves a large set of initiation factors (eIFs). Deregulation leads to altered gene expression, tumor formation and cancer. Translation initiation requires unwinding of secondary structures in the 5’-UTR of mRNAs by the DEAD-box helicase eIF4A, an RNA-dependent ATPase and ATP-dependent helicase. Together with the cap-binding protein eIF4E and the scaffold protein eIF4G, eIF4A forms the eIF4F complex that recognizes the 5’-cap and, assisted by eIF4B, recruits the 40S ribosomal subunit.

Critical Role for Cap-Independent c-MYC Translation in Progression of Multiple Myeloma.

Dysregulated c-myc is a determinant of multiple myeloma progression. Translation of c-myc can be achieved by an mTOR-mediated, cap-dependent mechanism or a cap-independent mechanism where a sequence in the 5'UTR of mRNA, termed the internal ribosome entry site (IRES), recruits the 40S ribosomal subunit. This mechanism requires the RNA-binding factor hnRNP A1 (A1) and becomes critical when cap-dependent translation is inhibited during endoplasmic reticulum (ER) stress. Thus, we studied the role of A1 and the myc IRES in myeloma biology. A1 expression correlated with enhanced c-myc expression in patient samples. Expression of A1 in multiple myeloma lines was mediated by c-myc itself, suggesting a positive feedback circuit where myc induces A1 and A1 enhances myc translation. We then deleted the A1 gene in a myc-driven murine myeloma model. A1-deleted multiple myeloma cells demonstrated downregulated myc expression and were inhibited in their growth in vivo. Decreased myc expression was due to reduced translational efficiency and depressed IRES activity. We also studied the J007 inhibitor, which prevents A1's interaction with the myc IRES. J007 inhibited myc translation and IRES activity and diminished myc expression in murine and human multiple myeloma lines as well as primary samples. J007 also inhibited tumor outgrowth in mice after subcutaneous or intravenous challenge and prevented osteolytic bone disease. When c-myc was ectopically reexpressed in A1-deleted multiple myeloma cells, tumor growth was reestablished. These results support the critical role of A1-dependent myc IRES translation in myeloma.

G-Quadruplex Regulation of VEGFA mRNA Translation by RBM4.

In eukaryotes, mRNAs translation is mainly mediated in a cap-dependent or cap-independent manner. The latter is primarily initiated at the internal ribosome entry site (IRES) in the 5′-UTR of mRNAs. It has been reported that the G-quadruplex structure (G4) in the IRES elements could regulate the IRES activity. We previously confirmed RBM4 (also known as LARK) as a G4-binding protein in human. In this study, to investigate whether RBM4 is involved in the regulation of the IRES activity by binding with the G4 structure within the IRES element, the IRES-A element in the 5′-UTR of vascular endothelial growth factor A (VEGFA) was constructed into a dicistronic reporter vector, psiCHECK2, and the effect of RBM4 on the IRES activity was tested in 293T cells. The results showed that the IRES insertion significantly increased the FLuc expression activity, indicating that this G4-containing IRES was active in 293T cells. When the G4 structure in the IRES was disrupted by base mutation, the IRES activity was significantly decreased. The IRES activity was notably increased when the cells were treated with G4 stabilizer PDS. EMSA results showed that RBM4 specifically bound the G4 structure in the IRES element. The knockdown of RBM4 substantially reduced the IRES activity, whereas over-expressing RBM4 increased the IRES activity. Taking all results together, we demonstrated that RBM4 promoted the mRNA translation of VEGFA gene by binding to the G4 structure in the IRES.

Identification of Bovine miRNAs with the Potential to Affect Human Gene Expression.

Milk and other products from large mammals have emerged during human evolution as an important source of nutrition. Recently, it has been recognized that exogenous miRNAs (mRNA inhibited RNA) contained in milk and other tissues of the mammalian body can enter the human body, which in turn have the ability to potentially regulate human metabolism by affecting gene expression. We studied for exogenous miRNAs from Bos taurus that are potentially contain miRNAs from milk and that could act postprandially as regulators of human gene expression. The interaction of 17,508 human genes with 1025 bta-miRNAs, including 245 raw milk miRNAs was studied. The milk bta-miR-151-5p, bta-miR-151-3p, bta-miRNA-320 each have 11 BSs (binding sites), and bta-miRNA-345-5p, bta-miRNA-614, bta-miRNA-1296b and bta-miRNA-149 has 12, 14, 15 and 26 BSs, respectively. The bta-miR-574-5p from cow’s milk had 209 human genes in mRNAs from one to 25 repeating BSs. We found 15 bta-miRNAs that have 100% complementarity to the mRNA of 13 human target genes. Another 12 miRNAs have BSs in the mRNA of 19 human genes with 98% complementarity. The bta-miR-11975, bta-miR-11976, and bta-miR-2885 BSs are located with the overlap of nucleotide sequences in the mRNA of human genes. Nucleotide sequences of BSs of these miRNAs in 5′UTR mRNA of human genes consisted of GCC repeats with a total length of 18 nucleotides (nt) in 18 genes, 21 nt in 11 genes, 24 nt in 14 genes, and 27–48 nt in nine genes. Nucleotide sequences of BSs of bta-miR-11975, bta-miR-11976, and bta-miR-2885 in CDS mRNA of human genes consisted of GCC repeats with a total length of 18 nt in 33 genes, 21 nt in 13 genes, 24 nt in nine genes, and 27–36 nt in 11 genes. These BSs encoded polyA or polyP peptides. In only one case, the polyR (SLC24A3 gene) was encoded. The possibility of regulating the expression of human genes by exogenous bovine miRNAs is discussed.

Ribosome ADP-ribosylation: A mechanism for maintaining protein homeostasis in cancers.

A recent study suggests that ribosome MARylation in cancers maintains proteostasis by reducing protein synthesis and preventing toxic protein aggregation. Mechanistically, NMNAT-2 is a cytosolic NAD+ synthase that supports the catalytic activity of PARP-16, which mediates ribosomal proteins MARylation. Ribosomal protein MARylation regulates polysomes assembly or function through the 3'-untranslated region (3'-UTR) stem-loop secondary structures in mRNAs, resulting in reduced protein synthesis and preventing toxic protein aggregation, thus supporting the growth of cancer cells during accelerated cell growth. When PARP-16 or NMNAT-2 is deleted, the stem-loop element in the 3'-UTRs of mRNAs increases polysome loading, enhances protein synthesis, promotes toxic protein aggregation, leading to inhibited cancer cell growth. Collectively, ribosome MARylation provides us with an exciting and scientific direction for us to understand cancers.

CRISPR-Mediated Knockout of Long 3' UTR mRNA Isoforms in mESC-Derived Neurons.

Alternative cleavage and polyadenylation (APA) is pervasive, occurring for more than 70% of human and mouse genes. Distal poly(A) site selection to generate longer 3′ UTR mRNA isoforms is prevalent in the nervous system, affecting thousands of genes. Here, we establish mouse embryonic stem cell (mESC)-derived neurons (mES-neurons) as a suitable system to study long 3′ UTR isoforms. RNA-seq analysis revealed that mES-neurons show widespread 3′ UTR lengthening that closely resembles APA patterns found in mouse cortex. mESCs are highly amenable to genetic manipulation. We present a method to eliminate long 3′ UTR isoform expression using CRISPR/Cas9 editing. This approach can lead to clones with the desired deletion within several weeks. We demonstrate this strategy on the Mprip gene as a proof-of-principle. To confirm loss of long 3′ UTR expression and the absence of cryptic poly(A) site usage stemming from the CRISPR deletion, we present a simple and cost-efficient targeted long-read RNA-sequencing strategy using the Oxford Nanopore Technologies platform. Using this method, we confirmed specific loss of the Mprip long 3′ UTR isoform. CRISPR gene editing of mESCs thus serves as a highly relevant platform for studying the molecular and cellular functions of long 3′ UTR mRNA isoforms.

TTP protects against acute liver failure by regulating CCL2 and CCL5 through m6A RNA methylation.

Tristetraprolin (TTP), an important immunosuppressive protein regulating mRNA decay through recognition of the AU-rich elements (AREs) within the 3′-UTRs of mRNAs, participates in the pathogenesis of liver diseases. However, whether TTP regulates mRNA stability through other mechanisms remains poorly understood. Here, we report that TTP was upregulated in acute liver failure (ALF), resulting in decreased mRNA stabilities of CCL2 and CCL5 through promotion of N6-methyladenosine (m6A) mRNA methylation. Overexpression of TTP could markedly ameliorate hepatic injury in vivo. TTP regulated the mRNA stabilization of CCL2 and CCL5. Interestingly, increased m6A methylation in CCL2 and CCL5 mRNAs promoted TTP-mediated RNA destabilization. Moreover, induction of TTP upregulated expression levels of WT1 associated protein, methyltransferase like 14, and YT521-B homology N6-methyladenosine RNA binding protein 2, which encode enzymes regulating m6A methylation, resulting in a global increase of m6A methylation and amelioration of liver injury due to enhanced degradation of CCL2 and CCL5. These findings suggest a potentially novel mechanism by which TTP modulates mRNA stabilities of CCL2 and CCL5 through m6A RNA methylation, which is involved in the pathogenesis of ALF.

Long noncoding RNA MIR22HG promotes Leydig cell apoptosis by acting as a competing endogenous RNA for microRNA-125a-5p that targets N-Myc downstream-regulated gene 2 in late-onset hypogonadism

Leydig cells (LCs) apoptosis is responsible for the deficiency of serum testosterone in Late-onset hypogonadism (LOH), while its specific mechanism is still unknown. This study focuses on the role of long noncoding RNA (lncRNA) MIR22HG in LC apoptosis and aims to elaborate its regulatory mechanism. MIR22HG was up-regulated in the testicular tissues of mice with LOH and H2O2-treated TM3 cells (mouse Leydig cell line). Interference of MIR22HG ameliorated cell apoptosis and upregulated miR-125a-5p expression in H2O2-treated TM3 cells. Then, the interaction between MIR22HG and miR-125a-5p was confirmed with RIP and RNA pull-down assay. Further study showed that miR-125a-5p downregulated N-Myc downstream-regulated gene 2 (NDRG2) expression by targeting its 3′-UTR of mRNA. What's more, MIR22HG overexpression aggravated cell apoptosis and reduced testosterone production in TM3 cells via miR-125a-5p/NDRG2 pathway. MIR22HG knockdown elevated testosterone levels in LOH mice. In conclusion, MIR22HG up-regulated NDRG2 expression through targeting miR-125a-5p, thus promoting LC apoptosis in LOH.Leydig cell apoptosis is responsible for the deficiency in serum testosterone in late-onset hypogonadism. This study reveals that lncRNA MIR22HG upregulates NDRG2 expression by targeting miR-125a-5p, thus promoting Leydig cell apoptosis in late-onset hypogonadism.

Crucial role of dead end gene for primordial germ cell survival in rice field eel (Monopterus albus)

The dead end gene has been identified as a essential factor for Primordial germ cells (PGCs) migration and survival in many species, but its role in Monopterus albus is unclear. In order to clarify the function of dead end gene in M.albus PGCs migration and survival, we first characterized the expression profile of M.albus dead end (Madnd) in developing embryos and various tissues. qRT-PCR revealed that Madnd transcripts were exclusively detected in gonad, including ovary, testis and ovotestis.Embryos injected with a Madnd morpholino (Madnd-MO) exhibited down-regulation of the vasa gene. Furthermore, the GFP signal show the PGCs migration in control group were injected with GFP-nanos3 3′-UTR mRNA for visualization, as described in a previous study, yet it was disappeared after embryos injected with Madnd-MO.Finally, we characterized the genomics sequence of the Madnd gene and designed five gRNAs for genome editing. Three gRNAs were selected for microinjection according to the results of in vitro tests. gRNAd1 was used for microinjection with the Cas9 protein and was confirmed to be effective. Our analysis in this study suggested that Madnd play a key role in PGCs migration and survival in M. albus. These data provide the basis for the production of fast-growing and reproductively M.albus sterile.

The prediction and expression of miR-203a-p and miR-29b* against DNMT3B as well as TNFAIP3 in melanoma

BackgroundThe development of melanoma could be derived by microRNA (miRNA)-mediated epigenetic regulation of tumor suppressor genes and oncogenes. miRNAs target some genes such as DNA methyltransferase 3-beta (DNMT3B) and consequently, DNMT3B could induce the expression of various genes like TNFAIP3. Therefore, the present study predicted the miRNAs targeting DNMT3B, and also evaluated the expression of TNFAIP3 in the melanoma cell lines. Materials and methodsAnalysis of gene suppression pathways and prediction of the targets of miRNAs including 3′UTR mRNA of DNMT3B gene was performed through various popular algorithms including miRanda, miRDB, miRWalk, RNAhybrid, PICTAR4, PICTAR5, PITA, RNA22, and Targetscan. Total RNA was extracted from the A375 cell line as melanoma cell and melanocyte cell line as a normal cell line. The expression of miRNAs, DNAMT3B, and TNFAIP3 was measured using Real-time PCR. ResultsMore than twenty miRNAs gained the highest scores by targeting the DNMT3B which among them hsa-miR-203a-p and hsa-miR-29b* were selected. A reduction in miRNAs and TNFAIP3 expression was observed according to the Real-time PCR (p-value 0.0323 and fold change 0.656). Also, the expression of DNMT3B increased significantly compared to the control group (p-value 0.0015 and fold change 3.482). ConclusionThe current study exhibited a decrease in the expression of TNFAIP3, miR-203a-pa-p, and miR-29b along with an increase in the expression of DNMT3B. These findings provided a helpful view of a new epigenetic-based approach for the early diagnosis of melanoma.

Introduction of an AU-rich Element into the 5’ UTR of mRNAs Enhances Protein Expression in Escherichia coli by S1 Protein and Hfq Protein

Introduction of an AU-rich Element into the 5’ UTR of mRNAs Enhances Protein Expression in Escherichia coli by S1 Protein and Hfq Protein

Bicc1 and Dicer regulate left-right patterning through post-transcriptional control of the Nodal inhibitor Dand5

Rotating cilia at the vertebrate left-right organizer (LRO) generate an asymmetric leftward flow, which is sensed by cells at the left LRO margin. Ciliary activity of the calcium channel Pkd2 is crucial for flow sensing. How this flow signal is further processed and relayed to the laterality-determining Nodal cascade in the left lateral plate mesoderm (LPM) is largely unknown. We previously showed that flow down-regulates mRNA expression of the Nodal inhibitor Dand5 in left sensory cells. De-repression of the co-expressed Nodal, complexed with the TGFß growth factor Gdf3, drives LPM Nodal cascade induction. Here, we show that post-transcriptional repression of dand5 is a central process in symmetry breaking of Xenopus, zebrafish and mouse. The RNA binding protein Bicc1 was identified as a post-transcriptional regulator of dand5 and gdf3 via their 3′-UTRs. Two distinct Bicc1 functions on dand5 mRNA were observed at pre- and post-flow stages, affecting mRNA stability or flow induced translational inhibition, respectively. To repress dand5, Bicc1 co-operates with Dicer1, placing both proteins in the process of flow sensing. Intriguingly, Bicc1 mediated translational repression of a dand5 3′-UTR mRNA reporter was responsive to pkd2, suggesting that a flow induced Pkd2 signal triggers Bicc1 mediated dand5 inhibition during symmetry breakage.

The Possible Mechanism of Physiological Adaptation to the Low-Se Diet and Its Health Risk in the Traditional Endemic Areas of Keshan Diseases.

Selenium is an essential trace element for humans and animals. As with oxygen and sulfur, etc., it belongs to the sixth main group of the periodic table of elements. Therefore, the corresponding amino acids, such as selenocysteine (Sec), serine (Ser), and cysteine (Cys), have similar spatial structure, physical, and chemical properties. In this review, we focus on the neglected but key role of serine in a possible mechanism of the physiological adaptation to Se-deficiency in human beings with an adequate intake of dietary protein: the insertion of Cys in place of Sec during the translation of selenoproteins dependent on the Sec insertion sequence element in the 3′UTR of mRNA at the UGA codon through a novel serine-dependent pathway for the de novo synthesis of the Cys-tRNA[Ser]Sec, similar to Sec-tRNA[Ser]Sec. We also discuss the important roles of serine in the metabolism of selenium directly or indirectly via GSH, and the maintenance of selenium homostasis regulated through the methylation modification of Sec-tRNA[Ser]Sec at the position 34U by SAM. Finally, we propose a hypothesis to explain why Keshan disease has gradually disappeared in China and predict the potential health risk of the human body in the physiological adaptation state of low selenium based on the results of animal experiments.

Efficiency of the synthetic self-splicing RiboJ ribozyme is robust to cis- and trans-changes in genetic background.

The expanding knowledge of the variety of synthetic genetic elements has enabled the construction of new and more efficient genetic circuits and yielded novel insights into molecular mechanisms. However, context dependence, in which interactions between cis‐ or trans‐genetic elements affect the behavior of these elements, can reduce their general applicability or predictability. Genetic insulators, which mitigate unintended context‐dependent cis‐interactions, have been used to address this issue. One of the most commonly used genetic insulators is a self‐splicing ribozyme called RiboJ, which can be used to decouple upstream 5′ UTR in mRNA from downstream sequences (e.g., open reading frames). Despite its general use as an insulator, there has been no systematic study quantifying the efficiency of RiboJ splicing or whether this autocatalytic activity is robust to trans‐ and cis‐genetic context. Here, we determine the robustness of RiboJ splicing in the genetic context of six widely divergent E. coli strains. We also check for possible cis‐effects by assessing two SNP versions close to the catalytic site of RiboJ. We show that mRNA molecules containing RiboJ are rapidly spliced even during rapid exponential growth and high levels of gene expression, with a mean efficiency of 98%. We also show that neither the cis‐ nor trans‐genetic context has a significant impact on RiboJ activity, suggesting this element is robust to both cis‐ and trans‐genetic changes.

CsrA regulation via binding to the base-pairing small RNA Spot 42.

The carbon storage regulator system and base-pairing small RNAs (sRNAs) represent two predominant modes of bacterial post-transcriptional regulation, which globally influence gene expression. Binding of CsrA protein to the 5' UTR or initial mRNA coding sequences can affect translation, RNA stability, and/or transcript elongation. Base-pairing sRNAs also regulate these processes, often requiring assistance from the RNA chaperone Hfq. Transcriptomics studies in Escherichia coli have identified many new CsrA targets, including Spot 42 and other base-pairing sRNAs. Spot 42 synthesis is repressed by cAMP-CRP, induced by the presence of glucose, and Spot 42 post-transcriptionally represses operons that facilitate metabolism of nonpreferred carbon sources. CsrA activity is also increased by glucose via effects on CsrA sRNA antagonists, CsrB/C. Here, we elucidate a mechanism wherein CsrA binds to and protects Spot 42 sRNA from RNase E-mediated cleavage. This protection leads to enhanced repression of srlA by Spot 42, a gene required for sorbitol uptake. A second, independent mechanism by which CsrA represses srlA is by binding to and inhibiting translation of srlM mRNA, encoding a transcriptional activator of srlA. Our findings demonstrate a novel means of regulation, by CsrA binding to a sRNA, and indicate that such interactions can help to shape complex bacterial regulatory circuitry.