Mechanism Of Translational Repression Research Articles

DECIPHERING THE ROLE OF EXPERIMENTALLY VALIDATED NICOTIANA TABACUM (TOBACCO) MIRNAS IN HUMAN HEALTH – A COMPUTATIONAL GENOMICS ASSESSMENT

Tobacco (Nicotiana tabacum) is considered as the tropical model plant for research especially for alkaloid like nicotine. One of the public health problems worldwide is harmful usage of tobacco that kills half of their consumers. On the other hand, Nicotiana tabacum was used as chief medicinal plants by native Americans, Amazonian and ancient Indians to cure poisonous reptiles’ bites and multiple diseases. MicroRNA (miRNA) is a prime gene regulator amongst the class of small-RNAs which binds with mRNA using translational repression or cleavage mechanism. Till the date, tobacco plant derived miRNAs were studied to check stress response in different biotic and abiotic condition and phylogenetic analysis, plant growth and development. Thus, cross-kingdom approach helps to understand the possible regulation as well as modulation in human health targeted by tobacco specific miRNAs. Tobacco derived miRNAs along with their targets were predicted and functionally annotated, pathway enrichment and disease association were studied in this study. Conclusively, we can report that N. tabacum miRNAs showed association with carcinoma and multiple neural, cardiac disorders.

Functional characterization of testis-brain RNA-binding protein, TB-RBP/Translin, in translational regulation

Testis-brain RNA-binding protein (TB-RBP/Translin) is known to contribute to the translational repression of a subset of haploid cell-specific mRNAs, including protamine 2 (Prm2) mRNA. Mutant mice lacking TB-RBP display abnormal spermatogenesis, despite normal male fertility. In this study, we carried out functional analysis of TB-RBP in mammalian cultured cells to understand the mechanism of translational repression by this RNA-binding protein. Although the amino acid sequence contained a eukaryotic translation initiation factor 4E (EIF4E)-recognition motif, TB-RBP failed to interact with EIF4E. In cultured cells, TB-RBP was unable to reduce the activity of luciferase encoded by a reporter mRNA carrying the 3’-untranslated region of Prm2. However, λΝ-BoxB tethering assay revealed that the complex of TB-RBP with its binding partner, Translin-associated factor X (TRAX), exhibits the ability to reduce the luciferase reporter activity by degrading the mRNA. These results suggest that TB-RBP may play a regulatory role in determining the sequence specificity of TRAX-catalyzed mRNA degradation.

Stress-Induced Translation Inhibition through Rapid Displacement of Scanning Initiation Factors

SummaryCellular responses to environmental stress are frequently mediated by RNA-binding proteins (RBPs). Here, we examined global RBP dynamics in Saccharomyces cerevisiae in response to glucose starvation and heat shock. Each stress induced rapid remodeling of the RNA-protein interactome without corresponding changes in RBP abundance. Consistent with general translation shutdown, ribosomal proteins contacting the mRNA showed decreased RNA association. Among translation components, RNA association was most reduced for initiation factors involved in 40S scanning (eukaryotic initiation factor 4A [eIF4A], eIF4B, and Ded1), indicating a common mechanism of translational repression. In unstressed cells, eIF4A, eIF4B, and Ded1 primarily targeted the 5′ ends of mRNAs. Following glucose withdrawal, 5′ binding was abolished within 30 s, explaining the rapid translation shutdown, but mRNAs remained stable. Heat shock induced progressive loss of 5′ RNA binding by initiation factors over ∼16 min and provoked mRNA degradation, particularly for translation-related factors, mediated by Xrn1. Taken together, these results reveal mechanisms underlying translational control of gene expression during stress.

Translation inhibition from a distance: The small RNA SgrS silences a ribosomal protein S1‐dependent enhancer

Many bacterial small RNAs (sRNAs) efficiently inhibit translation of target mRNAs by forming a duplex that sequesters the Shine-Dalgarno (SD) sequence or start codon and prevents formation of the translation initiation complex. There are a growing number of examples of sRNA-mRNA binding interactions distant from the SD region, but how these mediate translational regulation remains unclear. Our previous work in Escherichia coli and Salmonella identified a mechanism of translational repression of manY mRNA by the sRNA SgrS through a binding interaction upstream of the manY SD. Here, we report that SgrS forms a duplex with a uridine-rich translation-enhancing element in the manY 5' untranslated region. Notably, we show that the enhancer is ribosome-dependent and that the small ribosomal subunit protein S1 interacts with the enhancer to promote translation of manY. In collaboration with the chaperone protein Hfq, SgrS interferes with the interaction between the translation enhancer and ribosomal protein S1 to repress translation of manY mRNA. Since bacterial translation is often modulated by enhancer-like elements upstream of the SD, sRNA-mediated enhancer silencing could be a common mode of gene regulation.

RNase L Reprograms Translation by Widespread mRNA Turnover Escaped by Antiviral mRNAs.

In response to foreign and endogenous double-stranded RNA (dsRNA), protein kinase R (PKR) and ribonuclease L (RNase L) reprogram translation in mammalian cells. PKR inhibits translation initiation through eIF2α phosphorylation, which triggers stress granule (SG) formation and promotes translation of stress responsive mRNAs. The mechanisms of RNase L-driven translation repression, its contribution to SG assembly, and its regulation of dsRNA stress-induced mRNAs are unknown. We demonstrate that RNase L drives translational shut-off in response to dsRNA by promoting widespread turnover of mRNAs. This alters stress granule assembly and reprograms translation by allowing translation of mRNAs resistant to RNase L degradation, including numerous antiviral mRNAs such as interferon (IFN)-β. Individual cells differentially activate dsRNA responses revealing variation that can affect cellular outcomes. This identifies bulk mRNA degradation and the resistance of antiviral mRNAs as the mechanism by which RNase L reprograms translation in response to dsRNA.

The Plant microRNA Pathway: The Production and Action Stages.

Plant microRNAs are an endogenous class of small regulatory RNA central to the posttranscriptional regulation of gene expression in plant development and environmental stress adaptation or in response to pathogen challenge. The plant microRNA pathway is readily separated into two distinct stages: (1) the production stage, which is localized to the plant cell nucleus and where the microRNA small RNA is processed from a double-stranded RNA precursor transcript, and (2) the action stage, which is localized to the plant cell cytoplasm and where the mature microRNA small RNA is loaded into an effector complex and is used by the complex as a sequence specificity guide to direct expression repression of target genes harboring highly complementary microRNA target sequences. Historical research indicated that the plant microRNA pathway was a highly structured, almost linear pathway requiring a small set of core machinery proteins. However, contemporary research has demonstrated that the plant microRNA pathway is highly dynamic, and to allow for this flexibility, a large and highly functionally diverse set of machinery proteins is now known to be required. For example, recent research has shown that plant microRNAs can regulate target gene expression via a translational repression mechanism of RNA silencing in addition to the standard messenger RNA cleavage-based mechanism of RNA silencing: a mode of RNA silencing originally assigned to all plant microRNAs. Using Arabidopsis thaliana as our model system, here we report on both the core and auxiliary sets of machinery proteins now known to be required for both microRNA production and microRNA action in plants.

Inducible microRNA-590-5p inhibits host antiviral response by targeting the soluble interleukin-6 (IL6) receptor

MicroRNA (miR)-590-5p has been identified as an important regulator of some signaling pathways such as cell proliferation and tumorigenesis. However, little is known about its role during viral infection. Here, we report that miR-590-5p was significantly induced by various viruses and effectively potentiated virus replication in different viral infection systems. Furthermore, miR-590-5p substantially attenuated the virus-induced expression of type I and type III interferons (IFNs) and inflammatory cytokines, resulting in impaired downstream antiviral signaling. Interleukin-6 receptor (IL6R) was identified as a target of miR-590-5p. Interestingly, the role of miR-590-5p in virus-triggered signaling was abolished in IL6R knockout cells, and this could be rescued by restoring the expression of the soluble IL6R (sIL6R) but not the membrane-bound IL6R (mIL6R), suggesting that sIL6R is indispensable for miR-590-5p in modulating the host antiviral response. Furthermore, miR-590-5p down-regulated endogenous sIL6R and mIL6R expression through a translational repression mechanism. These findings thus uncover a previously uncharacterized role and the underlying mechanism of miR-590-5p in the innate immune response to viral infection.

TaMIR1119, a miRNA family member of wheat (Triticum aestivum), is essential in the regulation of plant drought tolerance

Through regulating target genes via the mechanisms of posttranscriptional cleavage or translational repression, plant miRNAs involve diverse biological processes associating with plant growth, development, and abiotic stress responses. In this study, we functionally characterized TaMIR1119, a miRNA family member of wheat (Triticum aestivum), in regulating the drought adaptive response of plants. TaMIR1119 putatively targets six genes categorized into the functional classes of transcriptional regulation, RNA and biochemical metabolism, trafficking, and oxidative stress defense. Upon simulated drought stress, the TaMIR1119 transcripts abundance in roots was drastically altered, showing to be upregulated gradually within a 48-h drought regime and that the drought-induced transcripts were gradually restored along with a 48-h recovery treatment. In contrast, most miRNA target genes displayed reverse expression patterns to TaMIR1119, exhibiting a downregulated expression pattern upon drought and whose reduced transcripts were re-elevated along with a normal recovery treatment. These expression analysis results indicated that TaMIR1119 responds to drought and regulates the target genes mainly through a cleavage mechanism. Under drought stress, the tobacco lines with TaMIR1119 overexpression behaved improved phenotypes, showing increased plant biomass, photosynthetic parameters, osmolyte accumulation, and enhanced antioxidant enzyme (AE) activities relative to wild type. Three AE genes, NtFeSOD, NtCAT1;3, and NtSOD2;1, encoding superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) proteins, respectively, showed upregulated expression in TaMIR1119 overexpression lines, suggesting that they are involved in the regulation of AE activities and contribution to the improved cellular reactive oxygen species (ROS) homeostasis in drought-challenged transgenic lines. Our results indicate that TaMIR1119 plays critical roles in regulating plant drought tolerance through transcriptionally regulating the target genes that modulate osmolyte accumulation, photosynthetic function, and improve cellular ROS homeostasis of plants.

P53 Regulates the Expression of LRP1 and Apoptosis through a Stress Intensity-Dependent MicroRNA Feedback Loop

SUMMARYUnderstanding how p53 activates certain gene programs and not others is critical. Here, we identify low-density lipoprotein receptor-related protein 1 (LRP1), a transmembrane endocytic receptor, as a p53 target gene. We show that, although LRP1 transcript expression is upregulated in response to both sub-lethal and lethal doses of p53-activating stress, LRP1 protein is only upregulated in response to sub-lethal stress. Interestingly, lethal doses of p53-activating stress inhibit LRP1 de novo translation through an miRNA-based translational repression mechanism. We show that the p53-regulated miRNAs miR-103 and miR-107 are significantly upregulated by lethal doses of stress, resulting in suppression of LRP1 translation and cell death. Our results define a negative feedback loop involving the p53-regulated coding gene LRP1 and p53-regulated miRNA genes. These findings provide mechanistic insight into the selective expression of p53 target genes in response to different stress intensities to elicit either cell survival or cell death.

MicroRNAs as biomarkers of kidney allograft injuries ischemia reperfusion and acute rejection

Renal transplantation provides a significant increase in life expectancy for patients with end-stage kidney diseases. Despite the unequivocal progresses made in the last decades, many grafts injuries still jeopardize graft function leading to its loss. Currently, the allograft biopsy is the gold standard for diagnosing such conditions. However, it has many limitations including risk and cost. Therefore, it is desirable to develop non-invasive biomarkers able to lead to the diagnosis of graft injuries. This is particularly true during the delayed graft function (DGF) period in which the functional parameters are not available. MicroRNAs regulate their target genes through mechanisms of translational repression and messenger RNA (mRNA) degradation. A single miRNA can regulate the expression of hundreds of mRNAs and proteins, deregulation of miRNAs can lead to disruption and suppression of genes that operate in intracellular signaling cascades leading to disease conditions. Technological advances have allowed the accurate detection of miRNAs in biological fluids providing either qualitative or quantitative results. Here we reviewed the literature in which miRNAs were analyzed as injury markers in renal transplantation mainly for contribute the diagnosis of acute rejection (AR). In cross sectional studies miRNAs were found to be useful markers of AR in renal transplant patients and their heightened expression by may became useful on aiding the diagnosis of AR during the DGF period.

Translational regulation by bacterial small RNAs via an unusual Hfq-dependent mechanism.

In bacteria, the canonical mechanism of translational repression by small RNAs (sRNAs) involves sRNA-mRNA base pairing that occludes the ribosome binding site (RBS), directly preventing translation. In this mechanism, the sRNA is the direct regulator, while the RNA chaperone Hfq plays a supporting role by stabilizing the sRNA. There are a few examples where the sRNA does not directly interfere with ribosome binding, yet translation of the target mRNA is still inhibited. Mechanistically, this non-canonical regulation by sRNAs is poorly understood. Our previous work demonstrated repression of the mannose transporter manX mRNA by the sRNA SgrS, but the regulatory mechanism was unknown. Here, we report that manX translation is controlled by a molecular role-reversal mechanism where Hfq, not the sRNA, is the direct repressor. Hfq binding adjacent to the manX RBS is required for sRNA-mediated translational repression. Translation of manX is also regulated by another sRNA, DicF, via the same non-canonical Hfq-dependent mechanism. Our results suggest that the sRNAs recruit Hfq to its binding site or stabilize the mRNA-Hfq complex. This work adds to the growing number of examples of diverse mechanisms of translational regulation by sRNAs in bacteria.

The human RNA-binding protein and E3 ligase MEX-3C binds the MEX-3–recognition element (MRE) motif with high affinity

MEX-3 is a K-homology (KH) domain-containing RNA-binding protein first identified as a translational repressor in Caenorhabditis elegans, and its four orthologs (MEX-3A-D) in human and mouse were subsequently found to have E3 ubiquitin ligase activity mediated by a RING domain and critical for RNA degradation. Current evidence implicates human MEX-3C in many essential biological processes and suggests a strong connection with immune diseases and carcinogenesis. The highly conserved dual KH domains in MEX-3 proteins enable RNA binding and are essential for the recognition of the 3'-UTR and post-transcriptional regulation of MEX-3 target transcripts. However, the molecular mechanisms of translational repression and the consensus RNA sequence recognized by the MEX-3C KH domain are unknown. Here, using X-ray crystallography and isothermal titration calorimetry, we investigated the RNA-binding activity and selectivity of human MEX-3C dual KH domains. Our high-resolution crystal structures of individual KH domains complexed with a noncanonical U-rich and a GA-rich RNA sequence revealed that the KH1/2 domains of human MEX-3C bound MRE10, a 10-mer RNA (5'-CAGAGUUUAG-3') consisting of an eight-nucleotide MEX-3-recognition element (MRE) motif, with high affinity. Of note, we also identified a consensus RNA motif recognized by human MEX-3C. The potential RNA-binding sites in the 3'-UTR of the human leukocyte antigen serotype (HLA-A2) mRNA were mapped with this RNA-binding motif and further confirmed by fluorescence polarization. The binding motif identified here will provide valuable information for future investigations of the functional pathways controlled by human MEX-3C and for predicting potential mRNAs regulated by this enzyme.

Translation repression via modulation of the cytoplasmic poly(A)-binding protein in the inflammatory response.

Gene expression is precisely regulated during the inflammatory response to control infection and limit the detrimental effects of inflammation. Here, we profiled global mRNA translation dynamics in the mouse primary macrophage-mediated inflammatory response and identified hundreds of differentially translated mRNAs. These mRNAs' 3'UTRs have enriched binding motifs for several RNA-binding proteins, which implies extensive translational regulatory networks. We characterized one such protein, Zfp36, as a translation repressor. Using primary macrophages from a Zfp36-V5 epitope tagged knock-in mouse generated by CRISPR/Cas9-mediated genome editing, we found that the endogenous Zfp36 directly interacts with the cytoplasmic poly(A)-binding protein. Importantly, this interaction is required for the translational repression of Zfp36's target mRNAs in resolving inflammation. Altogether, these results uncovered critical roles of translational regulations in controlling appropriate gene expression during the inflammatory response and revealed a new biologically relevant molecular mechanism of translational repression via modulating the cytoplasmic poly(A)-binding protein.

Translational repression by a miniature inverted-repeat transposable element in the 3\u2032 untranslated region

Transposable elements constitute a substantial portion of eukaryotic genomes and contribute to genomic variation, function, and evolution. Miniature inverted-repeat transposable elements (MITEs), as DNA transposons, are widely distributed in plant and animal genomes. Previous studies have suggested that retrotransposons act as translational regulators; however, it remains unknown how host mRNAs are influenced by DNA transposons. Here we report a translational repression mechanism mediated by a stowaway-like MITE (sMITE) embedded in the 3′-untranslated region (3′-UTR) of Ghd2, a member of the CCT (CONSTANS [CO], CO-LIKE and TIMING OF CAB1) gene family in rice. Ghd2 regulates important agronomic traits, including grain number, plant height and heading date. Interestingly, the translational repression of Ghd2 by the sMITE mainly relies on Dicer-like 3a (OsDCL3a). Furthermore, other MITEs in the 3′-UTRs of different rice genes exhibit a similar effect on translational repression, thus suggesting that MITEs may exert a general regulatory function at the translational level.

Function and Regulation of MicroRNAs and Their Potential as Biomarkers in Paediatric Liver Disease.

MicroRNAs (miRNAs) are short non-coding RNAs involved in biological and pathological processes of every cell type, including liver cells. Transcribed from specific genes, miRNA precursors are processed in the cytoplasm into mature miRNAs and as part of the RNA-induced silencing complex (RISC) complex binds to messenger RNA (mRNA) by imperfect complementarity. This leads to the regulation of gene expression at a post-transcriptional level. The function of a number of different miRNAs in fibrogenesis associated with the progression of chronic liver disease has recently been elucidated. Furthermore, miRNAs have been shown to be both disease-and tissue-specific and are stable in the circulation, which has led to increasing investigation on their utility as biomarkers for the diagnosis of chronic liver diseases, including those in children. Here, we review the current knowledge on the biogenesis of microRNA, the mechanisms of translational repression and the use of miRNA as circulatory biomarkers in chronic paediatric liver diseases including cystic fibrosis associated liver disease, biliary atresia and viral hepatitis B.

Upstream Open Reading Frames Located in the Leader of Protein Kinase Mζ mRNA Regulate Its Translation.

For protein synthesis that occurs locally in dendrites, the translational control mechanisms are much more important for neuronal functioning than the transcription levels. Here, we show that uORFs (upstream open reading frames) in the 5′ untranslated region (5′UTR) play a critical role in regulation of the translation of protein kinase Mζ (PKMζ). Elimination of these uORFs activates translation of the reporter protein in vitro and in primary cultures of rat hippocampal neurons. Using cell-free translation systems, we demonstrate that translational initiation complexes are formed only on uORFs. Further, we address the mechanism of translational repression of PKMζ translation, by uORFs. We observed an increase in translation of the reporter protein under the control of PKMζ leader in neuronal culture during non-specific activation by picrotoxin. We also show that such a mechanism is similar to the mechanism seen in cell stress, as application of sodium arsenite to neuron cultures induced translation of mRNA carrying PKMζ 5′UTR similarly to picrotoxin activation. Therefore, we suppose that phosphorylation of eIF2a, like in cell stress, is a main regulator of PKMζ translation. Altogether, our findings considerably extend our understanding of the role of uORF in regulation of PKMζ translation in activated neurons, important at early stages of LTP.

Enforced expression of hsa-miR-125a-3p in breast cancer cells potentiates docetaxel sensitivity via modulation of BRCA1 signaling

Epigenetic gene inactivation by microRNAs (miRNAs) plays a key role in malignant transformation, prevention of apoptosis, drug resistance and metastasis. It has been shown that miR-125a is down-regulated in HER2-amplified and HER2-overexpressing breast cancers (BCa), and this miRNA is believed to serve as an important tumor suppressor. miR-125a has two mature forms: hsa-miR-125a-3p and hsa-miR-125a-5p. However, the functional details of these miRNAs in BCa, particularly during pathogenesis of drug resistance, remain largely unexplored. Herein, we reported that hsa-miR-125a-3p expression was significantly reduced in chemoresistant BCa tissues and in experimentally established chemoresistant BCa cells. hsa-miR-125a-3p knockdown promoted cell proliferation and compromised docetaxel (Dox)-induced cell death, whereas overexpression of hsa-miR-125a-3p attenuated Dox chemoresistance in BCa cells. From a mechanistic standpoint, hsa-miR-125a-3p directly targeted 3’-untranslated regions (3′-UTRs) of breast cancer early onset gene 1 (BRCA1) and inhibits its protein expression via translational repression mechanism. In addition, suppression of BRCA1 expression by siRNA treatment effectively improved hsa-miR-125a-3p deficiency-triggered chemoresistance in BCa cells. Collectively, these findings suggest that hsa-miR-125a-3p may function as a tumor suppressor by regulating the BRCA1 signaling, and reintroduction of hsa-miR-125a-3p analogs could be a potential adjunct therapy for advanced/chemoresistant BCa.

The DDX6-4E-T interaction mediates translational repression and P-body assembly.

4E-Transporter binds eIF4E via its consensus sequence YXXXXLΦ, shared with eIF4G, and is a nucleocytoplasmic shuttling protein found enriched in P-(rocessing) bodies. 4E-T inhibits general protein synthesis by reducing available eIF4E levels. Recently, we showed that 4E-T bound to mRNA however represses its translation in an eIF4E-independent manner, and contributes to silencing of mRNAs targeted by miRNAs. Here, we address further the mechanism of translational repression by 4E-T by first identifying and delineating the interacting sites of its major partners by mass spectrometry and western blotting, including DDX6, UNR, unrip, PAT1B, LSM14A and CNOT4. Furthermore, we document novel binding between 4E-T partners including UNR-CNOT4 and unrip-LSM14A, altogether suggesting 4E-T nucleates a complex network of RNA-binding protein interactions. In functional assays, we demonstrate that joint deletion of two short conserved motifs that bind UNR and DDX6 relieves repression of 4E-T-bound mRNA, in part reliant on the 4E-T-DDX6-CNOT1 axis. We also show that the DDX6-4E-T interaction mediates miRNA-dependent translational repression and de novo P-body assembly, implying that translational repression and formation of new P-bodies are coupled processes. Altogether these findings considerably extend our understanding of the role of 4E-T in gene regulation, important in development and neurogenesis.

Genome-wide mapping of transcription start sites yields novel insights into the primary transcriptome of Pseudomonas putida.

The environmental bacterium Pseudomonas putida is an organism endowed with a versatile metabolism and stress tolerance traits that are desirable in an efficient production organism. In this work, differential RNA sequencing was used to investigate the primary transcriptome and RNA regulatory elements of P. putida strain KT2440. A total of 7937 putative transcription start sites (TSSs) were identified, where over two-thirds were located either on the opposite strand or internal to annotated genes. For TSSs associated with mRNAs, sequence analysis revealed a clear Shine-Dalgarno sequence but a lack of conserved overrepresented promoter motifs. These TSSs defined approximately 50 leaderless transcripts and an abundance of mRNAs with long leader regions of which 18 contain RNA regulatory elements from the Rfam database. The thiamine pyrophosphate riboswitch upstream of the thiC gene was examined using an in vivo assay with GFP-fusion vectors and shown to function via a translational repression mechanism. Furthermore, 56 novel intergenic small RNAs and 8 putative actuaton transcripts were detected, as well as 8 novel open reading frames (ORFs). This study illustrates how global mapping of TSSs can yield novel insights into the transcriptional features and RNA output of bacterial genomes.

CsrA Participates in a PNPase Autoregulatory Mechanism by Selectively Repressing Translation of pnp Transcripts That Have Been Previously Processed by RNase III and PNPase.

Csr is a conserved global regulatory system that represses or activates gene expression posttranscriptionally. CsrA of Escherichia coli is a homodimeric RNA binding protein that regulates transcription elongation, translation initiation, and mRNA stability by binding to the 5' untranslated leader or initial coding sequence of target transcripts. pnp mRNA, encoding the 3' to 5' exoribonuclease polynucleotide phosphorylase (PNPase), was previously identified as a CsrA target by transcriptome sequencing (RNA-seq). Previous studies also showed that RNase III and PNPase participate in a pnp autoregulatory mechanism in which RNase III cleavage of the untranslated leader, followed by PNPase degradation of the resulting 5' fragment, leads to pnp repression by an undefined translational repression mechanism. Here we demonstrate that CsrA binds to two sites in pnp leader RNA but only after the transcript is fully processed by RNase III and PNPase. In the absence of processing, both of the binding sites are sequestered in an RNA secondary structure, which prevents CsrA binding. The CsrA dimer bridges the upstream high-affinity site to the downstream site that overlaps the pnp Shine-Dalgarno sequence such that bound CsrA causes strong repression of pnp translation. CsrA-mediated translational repression also leads to a small increase in the pnp mRNA decay rate. Although CsrA has been shown to regulate translation and mRNA stability of numerous genes in a variety of organisms, this is the first example in which prior mRNA processing is required for CsrA-mediated regulation. CsrA protein represses translation of numerous mRNA targets, typically by binding to multiple sites in the untranslated leader region preceding the coding sequence. We found that CsrA represses translation of pnp by binding to two sites in the pnp leader transcript but only after it is processed by RNase III and PNPase. Processing by these two ribonucleases alters the mRNA secondary structure such that it becomes accessible to the ribosome for translation as well as to CsrA. As one of the CsrA binding sites overlaps the pnp ribosome binding site, bound CsrA prevents ribosome binding. This is the first example in which regulation by CsrA requires prior mRNA processing and should link pnp expression to conditions affecting CsrA activity.