Stem-loop RNA Research Articles

Visualizing Association of the Retroviral Gag Protein with Unspliced Viral RNA in the Nucleus.

Packaging of genomic RNA (gRNA) by retroviruses is essential for infectivity, yet the subcellular site of the initial interaction between the Gag polyprotein and gRNA remains poorly defined. Because retroviral particles are released from the plasma membrane, it was previously thought that Gag proteins initially bound to gRNA in the cytoplasm or at the plasma membrane. However, the Gag protein of the avian retrovirus Rous sarcoma virus (RSV) undergoes active nuclear trafficking, which is required for efficient gRNA encapsidation (L. Z. Scheifele, R. A. Garbitt, J. D. Rhoads, and L. J. Parent, Proc Natl Acad Sci U S A 99:3944-3949, 2002, https://doi.org/10.1073/pnas.062652199; R. Garbitt-Hirst, S. P. Kenney, and L. J. Parent, J Virol 83:6790-6797, 2009, https://doi.org/10.1128/JVI.00101-09). These results raise the intriguing possibility that the primary contact between Gag and gRNA might occur in the nucleus. To examine this possibility, we created a RSV proviral construct that includes 24 tandem repeats of MS2 RNA stem-loops, making it possible to track RSV viral RNA (vRNA) in live cells in which a fluorophore-conjugated MS2 coat protein is coexpressed. Using confocal microscopy, we observed that both wild-type Gag and a nuclear export mutant (Gag.L219A) colocalized with vRNA in the nucleus. In live-cell time-lapse images, the wild-type Gag protein trafficked together with vRNA as a single ribonucleoprotein (RNP) complex in the nucleoplasm near the nuclear periphery, appearing to traverse the nuclear envelope into the cytoplasm. Furthermore, biophysical imaging methods suggest that Gag and the unspliced vRNA physically interact in the nucleus. Taken together, these data suggest that RSV Gag binds unspliced vRNA to export it from the nucleus, possibly for packaging into virions as the viral genome.IMPORTANCE Retroviruses cause severe diseases in animals and humans, including cancer and acquired immunodeficiency syndromes. To propagate infection, retroviruses assemble new virus particles that contain viral proteins and unspliced vRNA to use as gRNA. Despite the critical requirement for gRNA packaging, the molecular mechanisms governing the identification and selection of gRNA by the Gag protein remain poorly understood. In this report, we demonstrate that the Rous sarcoma virus (RSV) Gag protein colocalizes with unspliced vRNA in the nucleus in the interchromatin space. Using live-cell confocal imaging, RSV Gag and unspliced vRNA were observed to move together from inside the nucleus across the nuclear envelope, suggesting that the Gag-gRNA complex initially forms in the nucleus and undergoes nuclear export into the cytoplasm as a viral ribonucleoprotein (vRNP) complex.

Structural Biology of the Enterovirus Replication-Linked 5'-Cloverleaf RNA and Associated Virus Proteins.

Although enteroviruses are associated with a wide variety of diseases and conditions, their mode of replication is well conserved. Their genome is carried as a single, positive-sense RNA strand. At the 5' end of the strand is an approximately 90-nucleotide self-complementary region called the 5' cloverleaf, or the oriL. This noncoding region serves as a platform upon which host and virus proteins, including the 3B, 3C, and 3D virus proteins, assemble in order to initiate replication of a negative-sense RNA strand. The negative strand in turn serves as a template for synthesis of multiple positive-sense RNA strands. Building on structural studies of individual RNA stem-loops, the structure of the intact 5' cloverleaf from rhinovirus has recently been determined via nuclear magnetic resonance/small-angle X-ray scattering (NMR/SAXS)-based methods, while structures have also been determined for enterovirus 3A, 3B, 3C, and 3D proteins. Analysis of these structures, together with structural and modeling studies of interactions between host and virus proteins and RNA, has begun to provide insight into the enterovirus replication mechanism and the potential to inhibit replication by blocking these interactions.

The construction and application of lentiviral overexpression vector of goat miR-204 in testis

The miRNA gene in DNA is first transcribed to Pri-miRNA, and then processed to Pre-miRNA, a stem-loop RNA segment (precursor) and further to miRNA which binds to mRNA by Dicer protein complex. It was confirmed that goat miR-204 could regulate the expressions of Sirt1 and the SSCs' (Spermatogonial Stem Cells) important genes Oct4 and Plzf, and inhibit the proliferation of dairy goat SSCs in vitro in our previous work. So, the research in vivo was needed next. In this study, the recombinant lentivirus vector pCDH-CMV-mir204-EF1-GreenPuro containing a goat chi-pri-mir-204 gene DNA segment was structured, and transfected into 293 T cells for packaged lentivirus, which then were injected into mouse seminiferous tubules. After 7 days, the goat miR-204 and the related genes such as Sirt1 and Plzf were detected in the mouse testis. This work laid a good foundation for further study of miR-204 biological function in vivo.

RNA Imaging with RNase-Inactivated Csy4 in Plants and Filamentous Fungi.

Subcellular localizations of RNAs can be imaged in vivo with genetically encoded reporters consisting of a sequence-specific RNA-binding protein (RBP) fused to a fluorescent protein. Several such reporter systems have been described based on RBPs that recognize RNA stem-loops. Here we describe RNA tagging for imaging with an inactive mutant of the bacterial endonuclease Csy4, which has a significantly higher affinity for its cognate stem-loop than alternative systems. This property allows for sensitive imaging with only few tandem copies of the target stem-loop inserted into the RNA of interest.

Intratumoral delivery of RIG-I agonist SLR14 induces robust antitumor responses.

Cytosolic nucleic acid-sensing pathways can be triggered to enhance immune response to cancer. In this study, we tested the antitumor activity of a unique RIG-I agonist, stem loop RNA (SLR) 14. In the immunogenic tumor models, we observed significant tumor growth delay and an extended survival in SLR14-treated mice. SLR14 also greatly improved antitumor efficacy of anti-PD1 antibody over single-agent treatment. SLR14 was mainly taken up by CD11b+ myeloid cells in the tumor microenvironment, and many genes associated with immune defense were significantly up-regulated after treatment, accompanied by increase in the number of CD8+ T lymphocytes, NK cells, and CD11b+ cells in SLR14-treated tumors. Strikingly, SLR14 dramatically inhibited nonimmunogenic B16 tumor growth, and the cured mice developed an immune memory. Furthermore, a systemic antitumor response was observed in both bilateral and tumor metastasis models. Collectively, our results demonstrate that SLR14 is a promising therapeutic RIG-I agonist for cancer treatment, either alone or in combination with existing immunotherapies.

A blue light receptor that mediates RNA binding and translational regulation.

Sensory photoreceptor proteins underpin light-dependent adaptations in nature and enable the optogenetic control of organismal behavior and physiology. We identified the bacterial light-oxygen-voltage (LOV) photoreceptor PAL that sequence-specifically binds short RNA stem loops with around 20 nM affinity in blue light and weaker than 1 μM in darkness. A crystal structure rationalizes the unusual receptor architecture of PAL with C-terminal LOV photosensor and N-terminal effector units. The light-activated PAL:RNA interaction can be harnessed to regulate gene expression at the RNA level as a function of light in both bacteria and mammalian cells. The present results elucidate a new signal-transduction paradigm in LOV receptors and conjoin RNA biology with optogenetic regulation, thereby paving the way towards hitherto inaccessible optoribogenetic modalities.

Relevance of the iron-responsive element (IRE) pseudotriloop structure for IRP1/2 binding and validation of IRE-like structures using the yeast three-hybrid system

Iron-responsive elements (IREs) are ~35-nucleotide (nt) stem-loop RNA structures located in 5′ or 3′ untranslated regions (UTRs) of mRNAs that mediate post-transcriptional regulation by their association with IRE-binding proteins (IRPs). IREs are characterized by their apical 6-nt loop motif 5’-CAGWGH-3′ (W = A or U and H = A, C or U), the so-called pseudotriloop, of which the loop nts C1 and G5 are paired, and the none-paired C between the two stem regions. In this study, the yeast three-hybrid (Y3H) system was used to investigate the relevance of the pseudotriloop structure of ferritin light chain (FTL) for the IRE-IRP interaction and the binding affinities between variant IRE(-like) structures and the two IRP isoforms, IRP1 and 2. Destabilization of the pseudotriloop structure by a G5-to-A mutation reduced binding of IRP1 and 2, while restoring the pseudotriloop conformation by the compensatory C1-to-U mutation, restored binding to both IRPs. In particular, IRP1 showed even stronger binding to the C1U-G5A mutant than to the wildtype FTL IRE. On the other hand, deletion of the bulged-out U6 of the pseudotriloop did not significantly affect its binding to either IRP1 or 2, but substitution with C particularly enhanced the binding to IRP1. In comparison to FTL IRE, IRE-like structures of 5′-aminolevulinate synthase 2 (ALAS2) and SLC40A1 (also known as ferroportin-1) showed similar or, in the case of endothelial PAS domain protein 1 (EPAS1) IRE, slightly weaker binding affinity to IRPs. SLC11A2 (a.k.a. divalent metal transporter-1) IRE exhibited relatively weak binding to IRP1 and medium binding to IRP2. Notably, the IRE-like structure of α-synuclein showed no detectable binding to either IRP under the conditions used in this Y3H assay. Our results indicate that Y3H can be used to characterize binding between IRPs and various IRE-like structures in vivo.

Enhancing the ligand efficiency of anti-HIV compounds targeting frameshift-stimulating RNA.

Ribosomal frameshifting, a process whereby a translating ribosome is diverted from one reading frame to another on a contiguous mRNA, is an important regulatory mechanism in biology and an opportunity for therapeutic intervention in several human diseases. In HIV, ribosomal frameshifting controls the ratio of Gag and Gag-Pol, two polyproteins critical to the HIV life cycle. We have previously reported compounds able to selectively bind an RNA stemloop within the Gag-Pol mRNA; these compounds alter the production of Gag-Pol in a manner consistent with increased frameshifting. Importantly, they also display antiretroviral activity in human T-cells. Here, we describe new compounds with significantly reduced molecular weight, but with substantially maintained affinity and anti-HIV activity. These results suggest that development of more "ligand efficient" enhancers of ribosomal frameshifting is an achievable goal.

Intratumoral delivery of RIG-I agonist induces robust anti-tumor immune responses

Abstract Recent studies have revealed that cytosolic nucleic acid-sensing pathways such as cGAS-STING and RIG-I-MAVS are involved in cancer biology. How to employ agents or agonists of these pathways to develop new approaches for cancer treatment is a promising direction. In this study, we synthesized a unique, binding-specific RIG-I agonist called Stem Loop RNA (SLR) 14 and evaluated its in vivo anti-tumor efficacy. When tumor volume reached 40–80 mm3, SLR14 was delivered intratumorally, every 2–3 days, for a total of 5–6 doses. Our results show that, after SLR14 treatment, B16 melanoma growth was dramatically blocked and the mice displayed long-term survival. We also observed a significant increase of tumor-infiltrating lymphocytes, and demonstrated that anti-tumor efficacy of SLR14 was T cell-dependent using RAG−/− mice and T cell depletion. Next, we tested anti-tumor efficacy of SLR14 in the mice with immunogenic tumor --YUMMER1.7 (melanoma) or MC38 (colon cancer). Similarly, we also observed a significant delay of tumor growth and extended mouse survival. Moreover, combination treatment with SLR14 and anti-PD1 in these mice led to more remarkable antitumor effects than single treatment. Lastly, we found that SLR14 was mainly taken up by CD45+CD11b+ leukocytes including neutrophils and myeloid cells, suggesting that anti-tumor efficacy of SLR14 is initiated through activation of the RIG-I pathway in innate cells within the tumor microenvironment. Taken together, our data demonstrate that synthetic RIG-I agonist SLR14 is a promising therapeutic agent for a broad spectrum of tumor subtypes. A better understanding of nucleic-acid sensing mechanisms within the tumor microenvironment may yield novel approaches for cancer immunotherapy.

Rapid RNA‐Protein Interaction Detection with RNA5.0 of HCMV

CMV is the most common cause of both congenital infection and post‐transplant infection worldwide. CMV infection can cause severe symptoms: pneumonia, encephalitis, and inflammation of vital organs. There is currently no vaccine for CMV. An understanding of how CMV progresses from its acute phase to persistence would illuminate features of the pathogen that could lead to novel treatments for the disease which block the virus from reaching latency.RNA5.0 is a lncRNA produced during CMV infection. Studies using the homologous RNA7.2 a mouse model has shown when the RNA is not expressed in vivo, CMV infection cannot reach its persistent phase. However, how it performs this function remains unclear. Our study hopes to help the field to move forward by expanding the limited knowledge about RNA5.0's function in viral persistence at a molecular level by purifying and identifying the proteins. We propose identifying proteins that bind to RNA5.0 with known cellular functions will allow us to narrow the speculation surrounding RNA5.0's function, as their functions are likely related.To purify these proteins, we will utilize the RaPID (RNA–protein interaction detection) method outlined in Ramanathan et al. 2018. In this novel approach, RNA stem loops flank RNA sequence of interest and recruits the RaPID fusion protein that biotinylates proteins proximal to inserted RNA. A streptavidin pulldown is used to capture biotinylated proteins for analysis by mass spectrometry (MS). Because the RaPID method has limited reach, in order to characterize all the proteins that bind to the 5kb lncRNA of CMV we will use 50 overlapping 150bp segments with 50bp overlaps.Support or Funding InformationFort Lewis College Undergraduate Research, Scholarship, and Creative Activity GrantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Targeting the Iron-Response Elements of the mRNAs for the Alzheimer's Amyloid Precursor Protein and Ferritin to Treat Acute Lead and Manganese Neurotoxicity.

The therapeutic value of inhibiting translation of the amyloid precursor protein (APP) offers the possibility to reduce neurotoxic amyloid formation, particularly in cases of familial Alzheimer’s disease (AD) caused by APP gene duplications (Dup–APP) and in aging Down syndrome individuals. APP mRNA translation inhibitors such as the anticholinesterase phenserine, and high throughput screened molecules, selectively inhibited the uniquely folded iron-response element (IRE) sequences in the 5’untranslated region (5’UTR) of APP mRNA and this class of drug continues to be tested in a clinical trial as an anti-amyloid treatment for AD. By contrast, in younger age groups, APP expression is not associated with amyloidosis, instead it acts solely as a neuroprotectant while facilitating cellular ferroportin-dependent iron efflux. We have reported that the environmental metallotoxins Lead (Pb) and manganese (Mn) cause neuronal death by interfering with IRE dependent translation of APP and ferritin. The loss of these iron homeostatic neuroprotectants thereby caused an embargo of iron (Fe) export from neurons as associated with excess unstored intracellular iron and the formation of toxic reactive oxidative species (ROS). We propose that APP 5’UTR directed translation activators can be employed therapeutically to protect neurons exposed to high acute Pb and/or Mn exposure. Certainly, high potency APP translation activators, exemplified by the Food and Drug Administration (FDA) pre-approved M1 muscarinic agonist AF102B and high throughput-screened APP 5’UTR translation activators, are available for drug development to treat acute toxicity caused by Pb/Mn exposure to neurons. We conclude that APP translation activators can be predicted to prevent acute metal toxicity to neurons by a mechanism related to the 5’UTR specific yohimbine which binds and targets the canonical IRE RNA stem loop as an H-ferritin translation activator.

The Solution Structure of FUS Bound to RNA Reveals a Bipartite Mode of RNA Recognition with Both Sequence and Shape Specificity

Fused in sarcoma (FUS) is an RNA binding protein involved in regulating many aspects of RNA processing and linked to several neurodegenerative diseases. Transcriptomics studies indicate that FUS binds a large variety of RNA motifs, suggesting that FUS RNA binding might be quite complex. Here, we present solution structures of FUS zinc finger (ZnF) and RNA recognition motif (RRM) domains bound to RNA. These structures show a bipartite binding mode of FUS comprising of sequence-specific recognition of a NGGU motif via the ZnF and an unusual shape recognition of a stem-loop RNA via the RRM. In addition, sequence-independent interactions via the RGG repeats significantly increase binding affinity and promote destabilization of structured RNA conformation, enabling additional binding. We further show that disruption of the RRM and ZnF domains abolishes FUS function in splicing. Altogether, our results rationalize why deciphering the RNA binding mode of FUS has been so challenging.

Polarizable force field for RNA based on the classical drude oscillator.

RNA molecules are highly dynamic and capable of adopting a wide range of complex, folded structures. The factors driving the folding and dynamics of these structures are dependent on a balance of base pairing, hydration, base stacking, ion interactions, and the conformational sampling of the 2'-hydroxyl group in the ribose sugar. The representation of these features is a challenge for empirical force fields used in molecular dynamics simulations. Toward meeting this challenge, the inclusion of explicit electronic polarization is important in accurately modeling RNA structure. In this work, we present a polarizable force field for RNA based on the classical Drude oscillator model, which represents electronic degrees of freedom via negatively charged particles attached to their parent atoms by harmonic springs. Beginning with parametrization against quantum mechanical base stacking interaction energy and conformational energy data, we have extended the Drude-2017 nucleic acid force field to include RNA. The conformational sampling of a range of RNA sequences were used to validate the force field, including canonical A-form RNA duplexes, stem-loops, and complex tertiary folds that bind multiple Mg2+ ions. Overall, the Drude-2017 RNA force field reproduces important properties of these structures, including the conformational sampling of the 2'-hydroxyl and key interactions with Mg2+ ions. © 2018 Wiley Periodicals, Inc.

Processive incorporation of multiple selenocysteine residues is driven by a novel feature of the selenocysteine insertion sequence

RNA stem loop structures have been frequently shown to regulate essential cellular processes. The selenocysteine insertion sequence (SECIS) element, found in the 3' UTRs of all selenoprotein mRNAs, is an example of such a structure, as it is required for the incorporation of the 21st amino acid, selenocysteine (Sec). Selenoprotein synthesis poses a mechanistic challenge because Sec is incorporated during translation in response to a stop codon (UGA). Although it is known that a SECIS-binding protein (SBP2) is required for Sec insertion, the mechanism of action remains elusive. Additional complexity is present in the synthesis of selenoprotein P (SELENOP), which is the only selenoprotein that contains multiple UGA codons and possesses two SECIS elements in its 3' UTR. Thus, full-length SELENOP synthesis requires processive Sec incorporation. Using zebrafish Selenop, in vitro translation assays, and 75Se labeling in HEK293 cells, we found here that processive Sec incorporation is an intrinsic property of the SECIS elements. Specifically, we identified critical features of SECIS elements that are required for processive Sec incorporation. A screen of the human SECIS elements revealed that most of these elements support processive Sec incorporation in vitro; however, we also found that the processivity of Sec incorporation into Selenop in cells is tightly regulated. We propose a model for processive Sec incorporation that involves differential recruitment of SECIS-binding proteins.

Regulation of Plant Microprocessor Function in Shaping microRNA Landscape.

MicroRNAs are small molecules (∼21 nucleotides long) that are key regulators of gene expression. They originate from long stem–loop RNAs as a product of cleavage by a protein complex called Microprocessor. The core components of the plant Microprocessor are the RNase type III enzyme Dicer-Like 1 (DCL1), the zinc finger protein Serrate (SE), and the double-stranded RNA binding protein Hyponastic Leaves 1 (HYL1). Microprocessor assembly and its processing of microRNA precursors have been reported to occur in discrete nuclear bodies called Dicing bodies. The accessibility of and modifications to Microprocessor components affect microRNA levels and may have dramatic consequences in plant development. Currently, numerous lines of evidence indicate that plant Microprocessor activity is tightly regulated. The cellular localization of HYL1 is dependent on a specific KETCH1 importin, and the E3 ubiquitin ligase COP1 indirectly protects HYL1 from degradation in a light-dependent manner. Furthermore, proper localization of HYL1 in Dicing bodies is regulated by MOS2. On the other hand, the Dicing body localization of DCL1 is regulated by NOT2b, which also interacts with SE in the nucleus. Post-translational modifications are substantial factors that contribute to protein functional diversity and provide a fine-tuning system for the regulation of protein activity. The phosphorylation status of HYL1 is crucial for its activity/stability and is a result of the interplay between kinases (MPK3 and SnRK2) and phosphatases (CPL1 and PP4). Additionally, MPK3 and SnRK2 are known to phosphorylate SE. Several other proteins (e.g., TGH, CDF2, SIC, and RCF3) that interact with Microprocessor have been found to influence its RNA-binding and processing activities. In this minireview, recent findings on the various modes of Microprocessor activity regulation are discussed.

Targeting RNA structure in SMN2 reverses spinal muscular atrophy molecular phenotypes

Modification of SMN2 exon 7 (E7) splicing is a validated therapeutic strategy against spinal muscular atrophy (SMA). However, a target-based approach to identify small-molecule E7 splicing modifiers has not been attempted, which could reveal novel therapies with improved mechanistic insight. Here, we chose as a target the stem-loop RNA structure TSL2, which overlaps with the 5′ splicing site of E7. A small-molecule TSL2-binding compound, homocarbonyltopsentin (PK4C9), was identified that increases E7 splicing to therapeutic levels and rescues downstream molecular alterations in SMA cells. High-resolution NMR combined with molecular modelling revealed that PK4C9 binds to pentaloop conformations of TSL2 and promotes a shift to triloop conformations that display enhanced E7 splicing. Collectively, our study validates TSL2 as a target for small-molecule drug discovery in SMA, identifies a novel mechanism of action for an E7 splicing modifier, and sets a precedent for other splicing-mediated diseases where RNA structure could be similarly targeted.

Substrate specificity of human MCPIP1 endoribonuclease

MCPIP1, also known as Regnase-1, is a ribonuclease crucial for regulation of stability of transcripts related to inflammatory processes. Here, we report that MCPIP1 acts as an endonuclease by degrading several stem-loop RNA structures and single-stranded RNAs. Our studies revealed cleavage sites present in the stem-loops derived from the 3′ untranslated region of the interleukin-6 transcript. Furthermore, MCPIP1 induced endonuclease cleavage at the loop motif of stem-loop structures. Additionally, we observed that MCPIP1 could cleave single-stranded RNA fragments. However, RNA substrates shorter than 6 nucleotides were not further affected by MCPIP1 nucleolytic activity. In this study, we also determined the dissociation constants of full-length MCPIP1D141N and its ribonuclease domain PIN D141N with twelve oligonucleotides substrates. The equilibrium binding constants (Kd) for MCPIP1D141N and the RNA targets were approximately 10 nM. Interestingly, we observed that the presence of a zinc finger in the PIN domain increases the affinity of this protein fragment to 25-nucleotide-long stem-loop RNA but not to shorter ones. Furthermore, size exclusion chromatography of the MCPIP1 and PIN proteins suggested that MCPIP1 undergoes homooligomerization during interaction with RNA substrates. Our results provide insight into the mechanism of MCPIP1 substrate recognition and its affinity towards various oligonucleotides.

Evolutionary plasticity of the NHL domain underlies distinct solutions to RNA recognition

RNA-binding proteins regulate all aspects of RNA metabolism. Their association with RNA is mediated by RNA-binding domains, of which many remain uncharacterized. A recently reported example is the NHL domain, found in prominent regulators of cellular plasticity like the C. elegans LIN-41. Here we employ an integrative approach to dissect the RNA specificity of LIN-41. Using computational analysis, structural biology, and in vivo studies in worms and human cells, we find that a positively charged pocket, specific to the NHL domain of LIN-41 and its homologs (collectively LIN41), recognizes a stem-loop RNA element, whose shape determines the binding specificity. Surprisingly, the mechanism of RNA recognition by LIN41 is drastically different from that of its more distant relative, the fly Brat. Our phylogenetic analysis suggests that this reflects a rapid evolution of the domain, presenting an interesting example of a conserved protein fold that acquired completely different solutions to RNA recognition.

High-resolution RNA 3'-ends mapping of bacterial Rho-dependent transcripts.

Transcription termination in bacteria can occur either via Rho-dependent or independent (intrinsic) mechanisms. Intrinsic terminators are composed of a stem-loop RNA structure followed by a uridine stretch and are known to terminate in a precise manner. In contrast, Rho-dependent terminators have more loosely defined characteristics and are thought to terminate in a diffuse manner. While transcripts ending in an intrinsic terminator are protected from 3′-5′ exonuclease digestion due to the stem-loop structure of the terminator, it remains unclear what protects Rho-dependent transcripts from being degraded. In this study, we mapped the exact steady-state RNA 3′ ends of hundreds of Escherichia coli genes terminated either by Rho-dependent or independent mechanisms. We found that transcripts generated from Rho-dependent termination have precise 3′-ends at steady state. These termini were localized immediately downstream of energetically stable stem-loop structures, which were not followed by uridine rich sequences. We provide evidence that these structures protect Rho-dependent transcripts from 3′-5′ exonucleases such as PNPase and RNase II, and present data localizing the Rho-utilization (rut) sites immediately downstream of these protective structures. This study represents the first extensive in-vivo map of exact RNA 3′-ends of Rho-dependent transcripts in E. coli.

SRSF3 recruits DROSHA to the basal junction of primary microRNAs.

The Microprocessor complex, consisting of an RNase III DROSHA and the DGCR8 dimer, cleaves primary microRNA transcripts (pri-miRNAs) to initiate microRNA (miRNA) maturation. Pri-miRNAs are stem–loop RNAs, and ∼79% of them contain at least one of the three major and conserved RNA motifs, UG, UGU, and CNNC. We recently demonstrated that the basal UG and apical UGU motifs of pri-miRNAs interact with DROSHA and DGCR8, respectively. They help orient Microprocessor on pri-miRNA in a proper direction in which DROSHA and DGCR8 localize to the basal and apical pri-miRNA junctions, respectively. In addition, CNNC, located at ∼17 nucleotides (nt) from the Microprocessor cleavage site, interacts with SRSF3 (SRp20) to stimulate Microprocessor to process pri-miRNAs. The mechanism underlying this stimulation, however, is unknown. In this study, we discovered that SRSF3 recruits DROSHA to the basal junction in a CNNC-dependent manner, thereby enhancing Microprocessor activity. Furthermore, by generating various pri-miRNA substrates containing CNNC at different locations, we demonstrated that such stimulation only occurs when CNNC is located at ∼17 nt from the Microprocessor cleavage site. Our findings reveal the molecular mechanism of SRSF3 in pri-miRNA processing and support the previously proposed explanation for the highly conserved position of CNNC in SRSF3-enhanced pri-miRNA processing.