System-wide Profiling of RNA-Binding Proteins Uncovers Key Regulators of Virus Infection.

The compendium of RNA-binding proteins (RBPs) has been greatly expanded by the development of RNA-interactome capture (RIC). However, it remained unknown if the complement of RBPs changes in response to environmental perturbations and whether these rearrangements are important. To answer these questions, we developed "comparative RIC" and applied it to cells challenged with an RNA virus called sindbis (SINV). Over 200 RBPs display differential interaction with RNA upon SINV infection. These alterations are mainly driven by the loss of cellular mRNAs and the emergence of viral RNA. RBPs stimulated by the infection redistribute to viral replication factories and regulate the capacity of the virus to infect. For example, ablation of XRN1 causes cells to be refractory to SINV, while GEMIN5 moonlights as a regulator of SINV gene expression. In summary, RNA availability controls RBP localization and function in SINV-infected cells.

TRIM25 inhibits influenza A virus infection, destabilizes viral mRNA, but is redundant for activating the RIG-I pathway.

The E3 ubiquitin ligase TRIM25 is a key factor in the innate immune response to RNA viruses. TRIM25 has been shown to play a role in the retinoic-acid-inducible gene-1 (RIG-I) pathway, which triggers expression of type 1 interferons upon viral infection. We and others have shown that TRIM25 is an RNA-binding protein; however, the role of TRIM25 RNA-binding in the innate immune response to RNA viruses is unclear. Here, we demonstrate that influenza A virus (IAV A/PR/8/34_NS1(R38A/K41A)) infection is inhibited by TRIM25. Surprisingly, previously identified RNA-binding deficient mutant TRIM25ΔRBD and E3 ubiquitin ligase mutant TRIM25ΔRING, which lack E3 ubiquitin ligase activity, still inhibited IAV replication. Furthermore, we show that in human-derived cultured cells, activation of the RIG-I/interferon type 1 pathway mediated by either an IAV-derived 5'-triphosphate RNA or by IAV itself does not require TRIM25 activity. Additionally, we present new evidence that instead of TRIM25 directly inhibiting IAV transcription it binds and destabilizes IAV mRNAs. Finally, we show that direct tethering of TRIM25 to RNA is sufficient to downregulate the targeted RNA. In summary, our results uncover a potential mechanism that TRIM25 uses to inhibit IAV infection and regulate RNA metabolism.

Evidence for a fragile X messenger ribonucleoprotein 1 (FMR1) mRNA gain-of-function toxicity mechanism contributing to the pathogenesis of fragile X-associated premature ovarian insufficiency.

Fragile X-associated premature ovarian insufficiency (FXPOI) is among a family of disorders caused by expansion of a CGG trinucleotide repeat sequence located in the 5' untranslated region (UTR) of the fragile X messenger ribonucleoprotein 1 (FMR1) gene on the X chromosome. Women with FXPOI have a depleted ovarian follicle population, resulting in amenorrhea, hypoestrogenism, and loss of fertility before the age of 40. FXPOI is caused by expansions of the CGG sequence to lengths between 55 and 200 repeats, known as a FMRI premutation, however the mechanism by which the premutation drives disease pathogenesis remains unclear. Two main hypotheses exist, which describe an mRNA toxic gain-of-function mechanism or a protein-based mechanism, where repeat-associated non-AUG (RAN) translation results in the production of an abnormal protein, called FMRpolyG. Here, we have developed an in vitro granulosa cell model of the FMR1 premutation by ectopically expressing CGG-repeat RNA and FMRpolyG protein. We show that expanded CGG-repeat RNA accumulated in intranuclear RNA structures, and these aggregates were able to cause significant granulosa cell death independent of FMRpolyG expression. Using an innovative RNA pulldown, mass spectrometry-based approach we have identified proteins that are specifically sequestered by CGG RNA aggregates in granulosa cells in vitro, and thus may be deregulated as consequence of this interaction. Furthermore, we have demonstrated reduced expression of three proteins identified via our RNA pulldown (FUS, PA2G4 and TRA2ß) in ovarian follicles in a FMR1 premutation mouse model. Collectively, these data provide evidence for the contribution of an mRNA gain-of-function mechanism to FXPOI disease biology.

RNA pull-down confocal nanoscanning (RP-CONA) detects quercetin as pri-miR-7/HuR interaction inhibitor that decreases α-synuclein levels.

RNA-protein interactions are central to all gene expression processes and contribute to a variety of human diseases. Therapeutic approaches targeting RNA-protein interactions have shown promising effects on some diseases that are previously regarded as 'incurable'. Here, we developed a fluorescent on-bead screening platform, RNA Pull-Down COnfocal NAnoscanning (RP-CONA), to identify RNA-protein interaction modulators in eukaryotic cell extracts. Using RP-CONA, we identified small molecules that disrupt the interaction between HuR, an inhibitor of brain-enriched miR-7 biogenesis, and the conserved terminal loop of pri-miR-7-1. Importantly, miR-7's primary target is an mRNA of α-synuclein, which contributes to the aetiology of Parkinson's disease. Our method identified a natural product quercetin as a molecule able to upregulate cellular miR-7 levels and downregulate the expression of α-synuclein. This opens up new therapeutic avenues towards treatment of Parkinson's disease as well as provides a novel methodology to search for modulators of RNA-protein interaction.

TRIM25 mutation (p.C168*), coding for an E3 ubiquitin ligase, is a cause of early-onset autosomal dominant dementia with amyloid load and parkinsonism.

INTRODUCTION: Patients with familial early-onset dementia (EOD) pose a unique opportunity for gene identification studies. METHODS: We present the phenotype and whole-exome sequencing (WES) study of an autosomal dominant EOD family. Candidate genes were examined in a set of dementia cases and controls (n = 3712). Western blotting was conducted of the wild-type and mutant protein of the final candidate. RESULTS: Age at disease onset was 60 years (range 56 to 63). The phenotype comprised mixed amnestic and behavioral features, and parkinsonism. Cerebrospinal fluid and plasma biomarkers, and a positron emission tomography amyloid study suggested Alzheimer's disease. WES and the segregation pattern pointed to a nonsense mutation in the TRIM25 gene (p.C168*), coding for an E3 ubiquitin ligase, which was absent in the cohorts studied. Protein studies supported a loss-of-function mechanism. DISCUSSION: This study supports a new physiopathological mechanism for brain amyloidosis. Furthermore, it extends the role of E3 ubiquitin ligases dysfunction in the development of neurodegenerative diseases. HIGHLIGHTS: A TRIM25 nonsense mutation (p.C168*) is associated with autosomal dominant early-onset dementia and parkinsonism with biomarkers suggestive of Alzheimer's disease. TRIM25 protein studies support that the mutation exerts its effect through loss of function. TRIM25, an E3 ubiquitin ligase, is known for its role in the innate immune response but this is the first report of association with neurodegeneration. The role of TRIM25 dysfunction in development of amyloidosis and neurodegeneration merits a new line of research.

Regulation of pri-miRNA processing by a long noncoding RNA transcribed from an ultraconserved region.

Noncoding RNAs (ncRNAs) control cellular programs by affecting protein-coding genes, but evidence increasingly points to their involvement in a network of ncRNA-ncRNA interactions. Here, we show that a long ncRNA, Uc.283+A, controls pri-miRNA processing. Regulation requires complementarity between the lower stem region of the pri-miR-195 transcript and an ultraconserved sequence in Uc.283+A, which prevents pri-miRNA cleavage by Drosha. Mutation of the site in either RNA molecule uncouples regulation in vivo and in vitro. We propose a model in which lower-stem strand invasion by Uc.283+A impairs microprocessor recognition and efficient pri-miRNA cropping. In addition to identifying a case of RNA-directed regulation of miRNA biogenesis, our study reveals regulatory networks involving different ncRNA classes of importance in cancer.

Post-transcriptional control of miRNA biogenesis.

MicroRNAs (miRNAs) are important regulators of gene expression that bind complementary target mRNAs and repress their expression. Precursor miRNA molecules undergo nuclear and cytoplasmic processing events, carried out by the endoribonucleases DROSHA and DICER, respectively, to produce mature miRNAs that are loaded onto the RISC (RNA-induced silencing complex) to exert their biological function. Regulation of mature miRNA levels is critical in development, differentiation, and disease, as demonstrated by multiple levels of control during their biogenesis cascade. Here, we will focus on post-transcriptional mechanisms and will discuss the impact of cis-acting sequences in precursor miRNAs, as well as trans-acting factors that bind to these precursors and influence their processing. In particular, we will highlight the role of general RNA-binding proteins (RBPs) as factors that control the processing of specific miRNAs, revealing a complex layer of regulation in miRNA production and function.

Tissue-specific control of brain-enriched miR-7 biogenesis.

MicroRNA (miRNA) biogenesis is a highly regulated process in eukaryotic cells. Several mature miRNAs exhibit a tissue-specific pattern of expression without an apparent tissue-specific pattern for their corresponding primary transcripts. This discrepancy is suggestive of post-transcriptional regulation of miRNA abundance. Here, we demonstrate that the brain-enriched expression of miR-7, which is processed from the ubiquitous hnRNP K pre-mRNA transcript, is achieved by inhibition of its biogenesis in nonbrain cells in both human and mouse systems. Using stable isotope labeling by amino acids in cell culture (SILAC) mass spectrometry combined with RNase-assisted RNA pull-down, we identified Musashi homolog 2 (MSI2) and Hu antigen R (HuR) proteins as inhibitors of miR-7 processing in nonneural cells. This is achieved through HuR-mediated binding of MSI2 to the conserved terminal loop of pri-miR-7. Footprinting and electrophoretic gel mobility shift analysis (EMSA) provide further evidence for a direct interaction between pri-miR-7-1 and the HuR/MSI2 complex, resulting in stabilization of the pri-miR-7-1 structure. We also confirmed the physiological relevance of this inhibitory mechanism in a neuronal differentiation system using human SH-SY5Y cells. Finally, we show elevated levels of miR-7 in selected tissues from MSI2 knockout (KO) mice without apparent changes in the abundance of the pri-miR-7 transcript. Altogether, our data provide the first insight into the regulation of brain-enriched miRNA processing by defined tissue-specific factors.

Quantitative identification of proteins that influence miRNA biogenesis by RNA pull-down-SILAC mass spectrometry (RP-SMS).

RNA-binding proteins mediate and control gene expression. As some examples, they regulate pre-mRNA synthesis and processing; mRNA localisation, translation and decay; and microRNA (miRNA) biogenesis and function. Here, we present a detailed protocol for RNA pull-down coupled to stable isotope labelling by amino acids in cell culture (SILAC) mass spectrometry (RP-SMS) that enables quantitative, fast and specific detection of RNA-binding proteins that regulate miRNA biogenesis. In general, this method allows for the identification of RNA-protein complexes formed using in vitro or chemically synthesized RNAs and protein extracts derived from cultured cells.

RNA-Targeted Therapies and High-Throughput Screening Methods.

RNA-binding proteins (RBPs) are involved in regulating all aspects of RNA metabolism, including processing, transport, translation, and degradation. Dysregulation of RNA metabolism is linked to a plethora of diseases, such as cancer, neurodegenerative diseases, and neuromuscular disorders. Recent years have seen a dramatic shift in the knowledge base, with RNA increasingly being recognised as an attractive target for precision medicine therapies. In this article, we are going to review current RNA-targeted therapies. Furthermore, we will scrutinise a range of drug discoveries targeting protein-RNA interactions. In particular, we will focus on the interplay between Lin28 and let-7, splicing regulatory proteins and survival motor neuron (SMN) pre-mRNA, as well as HuR, Musashi, proteins and their RNA targets. We will highlight the mechanisms RBPs utilise to modulate RNA metabolism and discuss current high-throughput screening strategies. This review provides evidence that we are entering a new era of RNA-targeted medicine.

Lin28a uses distinct mechanisms of binding to RNA and affects miRNA levels positively and negatively.

Lin28a inhibits the biogenesis of let-7 miRNAs by triggering the polyuridylation and degradation of their precursors by terminal uridylyltransferases TUT4/7 and 3'-5' exoribonuclease Dis3l2, respectively. Previously, we showed that Lin28a also controls the production of neuro-specific miRNA-9 via a polyuridylation-independent mechanism. Here we reveal that the sequences and structural characteristics of pre-let-7 and pre-miRNA-9 are eliciting two distinct modes of binding to Lin28a. We present evidence that Dis3l2 controls miRNA-9 production. Finally, we show that the constitutive expression of untagged Lin28a during neuronal differentiation in vitro positively and negatively affects numerous other miRNAs. Our findings shed light on the role of Lin28a in differentiating cells and on the ways in which one RNA-binding protein can perform multiple roles in the regulation of RNA processing.

Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection.

Delivery of DNA to cells and its subsequent integration into the host genome is a fundamental task in molecular biology, biotechnology and gene therapy. Here we describe an IP-free one-step method that enables stable genome integration into either prokaryotic or eukaryotic cells. A synthetic mariner transposon is generated by flanking a DNA sequence with short inverted repeats. When purified recombinant Mos1 or Mboumar-9 transposase is co-transfected with transposon-containing plasmid DNA, it penetrates prokaryotic or eukaryotic cells and integrates the target DNA into the genome. In vivo integrations by purified transposase can be achieved by electroporation, chemical transfection or Lipofection of the transposase:DNA mixture, in contrast to other published transposon-based protocols which require electroporation or microinjection. As in other transposome systems, no helper plasmids are required since transposases are not expressed inside the host cells, thus leading to generation of stable cell lines. Since it does not require electroporation or microinjection, this tool has the potential to be applied for automated high-throughput creation of libraries of random integrants for purposes including gene knock-out libraries, screening for optimal integration positions or safe genome locations in different organisms, selection of the highest production of valuable compounds for biotechnology, and sequencing.

RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain and is required for ubiquitination.

BACKGROUND: TRIM25 is a novel RNA-binding protein and a member of the Tripartite Motif (TRIM) family of E3 ubiquitin ligases, which plays a pivotal role in the innate immune response. However, there is scarce knowledge about its RNA-related roles in cell biology. Furthermore, its RNA-binding domain has not been characterized. RESULTS: Here, we reveal that the RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain, which we postulate to be a novel RNA-binding domain. Using CLIP-seq and SILAC-based co-immunoprecipitation assays, we uncover TRIM25's endogenous RNA targets and protein binding partners. We demonstrate that TRIM25 controls the levels of Zinc Finger Antiviral Protein (ZAP). Finally, we show that the RNA-binding activity of TRIM25 is important for its ubiquitin ligase activity towards itself (autoubiquitination) and its physiologically relevant target ZAP. CONCLUSIONS: Our results suggest that many other proteins with the PRY/SPRY domain could have yet uncharacterized RNA-binding potential. Together, our data reveal new insights into the molecular roles and characteristics of RNA-binding E3 ubiquitin ligases and demonstrate that RNA could be an essential factor in their enzymatic activity.

Hormonal regulation of microRNA biogenesis.

In this issue of Molecular Cell, Yamagata et al. (2009) provide insight into the complex posttranscriptional regulation of miRNA biogenesis by showing that the processing of a subset of miRNAs is inhibited by the estrogen receptor.

The role of Trim25 in development, disease and RNA metabolism.

Trim25 is a member of the tripartite motif family of E3 ubiquitin ligases. It plays major roles in innate immunity and defence against viral infection, control of cell proliferation and migration of cancer cells. Recent work identified Trim25 as being able to bind to RNA and to regulate Lin28a-mediated uridylation of pre-let-7. Here we review the current knowledge of the role of Trim25 in development, disease and RNA metabolism.

Posttranscriptional regulation of miRNAs harboring conserved terminal loops.

We recently found that hnRNP A1, a protein implicated in many aspects of RNA processing, acts as an auxiliary factor for the Drosha-mediated processing of a microRNA precursor, pri-miR-18a. Here, we provide the mechanism by which hnRNP A1 regulates this event. We show that hnRNP A1 binds to the loop of pri-miR-18a and induces a relaxation at the stem, creating a more favorable cleavage site for Drosha. We found that approximately 14% of all pri-miRNAs have highly conserved loops, which we predict act as landing pads for trans-acting factors influencing miRNA processing. In agreement, we show that 2'O-methyl oligonucleotides targeting conserved loops (LooptomiRs) abolish miRNA processing in vitro. Furthermore, we present evidence to support an essential role of conserved loops for pri-miRNA processing. Altogether, these data suggest the existence of auxiliary factors for the processing of specific miRNAs, revealing an additional level of complexity for the regulation of miRNA biogenesis.

The splicing factor SF2/ASF regulates translation initiation by enhancing phosphorylation of 4E-BP1.

The SR protein SF2/ASF has been initially characterized as a splicing factor but has also been shown to mediate postsplicing activities such as mRNA export and translation. Here we demonstrate that SF2/ASF promotes translation initiation of bound mRNAs and that this activity requires the presence of the cytoplasmic cap-binding protein eIF4E. SF2/ASF promotes translation initiation by suppressing the activity of 4E-BP, a competitive inhibitor of cap-dependent translation. This activity is mediated by interactions of SF2/ASF with both mTOR and the phosphatase PP2A, two key regulators of 4E-BP phosphorylation. These findings suggest the model whereby SF2/ASF functions as an adaptor protein to recruit the signaling molecules responsible for regulation of cap-dependent translation of specific mRNAs. Taken together, these data suggest a novel mechanism for the activation of translation initiation of a subset of mRNAs bound by the shuttling protein SF2/ASF.

PaperClip: rapid multi-part DNA assembly from existing libraries.

Assembly of DNA 'parts' to create larger constructs is an essential enabling technique for bioengineering and synthetic biology. Here we describe a simple method, PaperClip, which allows flexible assembly of multiple DNA parts from currently existing libraries cloned in any vector. No restriction enzymes, mutagenesis of internal restriction sites, or reamplification to add end homology are required. Order of assembly is directed by double stranded oligonucleotides-'Clips'. Clips are formed by ligation of pairs of oligonucleotides corresponding to the ends of each part. PaperClip assembly can be performed by polymerase chain reaction or by cell extract-mediated recombination. Once multi-use Clips have been prepared, assembly of at least six DNA parts in any order can be accomplished with high efficiency within several hours.

Editing independent effects of ADARs on the miRNA/siRNA pathways.

Adenosine deaminases acting on RNA (ADARs) are best known for altering the coding sequences of mRNA through RNA editing, as in the GluR-B Q/R site. ADARs have also been shown to affect RNA interference (RNAi) and microRNA processing by deamination of specific adenosines to inosine. Here, we show that ADAR proteins can affect RNA processing independently of their enzymatic activity. We show that ADAR2 can modulate the processing of mir-376a2 independently of catalytic RNA editing activity. In addition, in a Drosophila assay for RNAi deaminase-inactive ADAR1 inhibits RNAi through the siRNA pathway. These results imply that ADAR1 and ADAR2 have biological functions as RNA-binding proteins that extend beyond editing per se and that even genomically encoded ADARs that are catalytically inactive may have such functions.

miRNAs in development and pathogenesis of the nervous system.

The human nervous system expresses approximately 70% of all miRNAs (microRNAs). Changing levels of certain ubiquitous and brain-specific miRNAs shape the development and function of the nervous system. It is becoming clear that misexpression of some miRNAs can contribute towards neurodevelopmental disorders. In the present article, we review the current knowledge of the role of miRNAs in development and pathogenesis of the nervous system.