AGO HITS-CLIP reveals distinct miRNA regulation of white and brown adipose tissue identity.

MicroRNAs (miRNAs) are short, noncoding RNAs that associate with Argonaute (AGO) to influence mRNA stability and translation, thereby regulating cellular determination and phenotype. While several individual miRNAs have been shown to control adipocyte function, including energy storage in white fat and energy dissipation in brown fat, a comprehensive analysis of miRNA activity in these tissues has not been performed. We used high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP) to comprehensively characterize the network of high-confidence, in vivo mRNA:miRNA interactions across white and brown fat, revealing >20,000 unique AGO binding sites. When coupled with miRNA and mRNA sequencing, we found an inverse correlation between depot-enriched miRNAs and their targets. To illustrate the functionality of our HITS-CLIP data set in identifying specific miRNA:mRNA interactions, we show that miR-29 is a novel regulator of leptin, an adipocyte-derived hormone that coordinates food intake and energy homeostasis. Two independent miR-29 binding sites in the leptin 3' UTR were validated using luciferase assays, and miR-29 gain and loss of function modulated leptin mRNA and protein secretion in primary adipocytes. This work represents the only experimentally generated miRNA targetome in adipose tissue and identifies multiple regulatory pathways that may specify the unique identities of white and brown fat.

An RNA-binding protein, Qki5, regulates embryonic neural stem cells through pre-mRNA processing in cell adhesion signaling.

Cell type-specific transcriptomes are enabled by the action of multiple regulators, which are frequently expressed within restricted tissue regions. In the present study, we identify one such regulator, Quaking 5 (Qki5), as an RNA-binding protein (RNABP) that is expressed in early embryonic neural stem cells and subsequently down-regulated during neurogenesis. mRNA sequencing analysis in neural stem cell culture indicates that Qki proteins play supporting roles in the neural stem cell transcriptome and various forms of mRNA processing that may result from regionally restricted expression and subcellular localization. Also, our in utero electroporation gain-of-function study suggests that the nuclear-type Qki isoform Qki5 supports the neural stem cell state. We next performed in vivo transcriptome-wide protein-RNA interaction mapping to search for direct targets of Qki5 and elucidate how Qki5 regulates neural stem cell function. Combined with our transcriptome analysis, this mapping analysis yielded a bona fide map of Qki5-RNA interaction at single-nucleotide resolution, the identification of 892 Qki5 direct target genes, and an accurate Qki5-dependent alternative splicing rule in the developing brain. Last, our target gene list provides the first compelling evidence that Qki5 is associated with specific biological events; namely, cell-cell adhesion. This prediction was confirmed by histological analysis of mice in which Qki proteins were genetically ablated, which revealed disruption of the apical surface of the lateral wall in the developing brain. These data collectively indicate that Qki5 regulates communication between neural stem cells by mediating numerous RNA processing events and suggest new links between splicing regulation and neural stem cell states.

HITS-CLIP analysis of human ALKBH8 reveals interactions with fully processed substrate tRNAs and with specific noncoding RNAs.

Transfer RNAs acquire a large plethora of chemical modifications. Among those, modifications of the anticodon loop play important roles in translational fidelity and tRNA stability. Four human wobble U-containing tRNAs obtain 5-methoxycarbonylmethyluridine (mcm5U34) or 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U34), which play a role in decoding. This mark involves a cascade of enzymatic activities. The last step is mediated by alkylation repair homolog 8 (ALKBH8). In this study, we performed a transcriptome-wide analysis of the repertoire of ALKBH8 RNA targets. Using a combination of HITS-CLIP and RIP-seq analyses, we uncover ALKBH8-bound RNAs. We show that ALKBH8 targets fully processed and CCA modified tRNAs. Our analyses uncovered the previously known set of wobble U-containing tRNAs. In addition, both our approaches revealed ALKBH8 binding to several other types of noncoding RNAs, in particular C/D box snoRNAs.

Next-generation sequencing: A new avenue to understand viral RNA-protein interactions.

The genomes of RNA viruses present an astonishing source of both sequence and structural diversity. From intracellular viral RNA-host interfaces to interactions between the RNA genome and structural proteins in virus particles themselves, almost the entire viral lifecycle is accompanied by a myriad of RNA-protein interactions that are required to fulfill their replicative potential. It is therefore important to characterize such rich and dynamic collections of viral RNA-protein interactions to understand virus evolution and their adaptation to their hosts and environment. Recent advances in next-generation sequencing technologies have allowed the characterization of viral RNA-protein interactions, including both transient and conserved interactions, where molecular and structural approaches have fallen short. In this review, we will provide a methodological overview of the high-throughput techniques used to study viral RNA-protein interactions, their biochemical mechanisms, and how they evolved from classical methods as well as one another. We will discuss how different techniques have fueled virus research to characterize how viral RNA and proteins interact, both locally and on a global scale. Finally, we will present examples on how these techniques influence the studies of clinically important pathogens such as HIV-1 and SARS-CoV-2.

Local changes in viral RNA sequence drive global changes in influenza nucleoprotein binding.

The genome of influenza A viruses (IAV) consists of eight negative-sense RNA segments that are coated by viral nucleoprotein (NP). Until recently, it was assumed that NP binds viral genomic RNA (vRNA) uniformly along the entire segment. However, genome-wide studies have revised the original model in that NP instead binds preferentially to certain regions of vRNA, while others are depleted for NP binding. Even strains with high sequence similarity exhibit distinct NP-binding profiles. Thus, it remains unknown how NP-binding specificity to vRNA is established. Here we introduced nucleotide changes to vRNA to examine whether primary sequence can affect NP binding. Our findings demonstrate that NP binding is indeed susceptible to sequence alterations, as NP peaks can be lost or appear de novo at mutated sites. Unexpectedly, nucleotide changes not only affect NP binding locally at the site of mutation, but also impact NP binding at distal regions that have not been modified. Taken together, our results suggest that NP binding is not regulated by primary sequence alone, but that a network formed by multiple segments governs the deposition of NP on vRNA.

LIN28A binds to meiotic gene transcripts and modulates their translation in male germ cells.

The RNA-binding protein LIN28A is required for maintaining tissue homeostasis, including in the reproductive system, but the underlying mechanisms on how LIN28A regulates germline progenitors remain unclear. Here, we dissected LIN28A-binding targets using high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP) in the mouse testes. LIN28A preferentially binds to mRNA coding sequence (CDS) or 3'UTR regions at sites enriched with GGAG(A) sequences. Further investigation of Lin28a-null mouse testes indicated that meiosis-associated mRNAs bound by LIN28A were differentially expressed. Next, ribosome profiling revealed that the mRNA levels of these targets were significantly reduced in the polysome fractions, and their protein expression levels decreased, in Lin28a-null mouse testes, even when meiotic arrest in the null mouse testes was not apparent. Collectively, these findings provide a set of LIN28A-regulated target mRNAs, and show that LIN28A binding might be a mechanism through which LIN28A acts to regulate undifferentiated spermatogonia fates and male fertility in mammals.

Star-PAP RNA Binding Landscape Reveals Novel Role of Star-PAP in mRNA Metabolism That Requires RBM10-RNA Association.

Star-PAP is a non-canonical poly(A) polymerase that selects mRNA targets for polyadenylation. Yet, genome-wide direct Star-PAP targets or the mechanism of specific mRNA recognition is still vague. Here, we employ HITS-CLIP to map the cellular Star-PAP binding landscape and the mechanism of global Star-PAP mRNA association. We show a transcriptome-wide association of Star-PAP that is diminished on Star-PAP depletion. Consistent with its role in the 3'-UTR processing, we observed a high association of Star-PAP at the 3'-UTR region. Strikingly, there is an enrichment of Star-PAP at the coding region exons (CDS) in 42% of target mRNAs. We demonstrate that Star-PAP binding de-stabilises these mRNAs indicating a new role of Star-PAP in mRNA metabolism. Comparison with earlier microarray data reveals that while UTR-associated transcripts are down-regulated, CDS-associated mRNAs are largely up-regulated on Star-PAP depletion. Strikingly, the knockdown of a Star-PAP coregulator RBM10 resulted in a global loss of Star-PAP association on target mRNAs. Consistently, RBM10 depletion compromises 3'-end processing of a set of Star-PAP target mRNAs, while regulating stability/turnover of a different set of mRNAs. Our results establish a global profile of Star-PAP mRNA association and a novel role of Star-PAP in the mRNA metabolism that requires RBM10-mRNA association in the cell.

Identification and removal of sequencing artifacts produced by mispriming during reverse transcription in multiple RNA-seq technologies.

The quality of RNA sequencing data relies on specific priming by the primer used for reverse transcription (RT-primer). Nonspecific annealing of the RT-primer to the RNA template can generate reads with incorrect cDNA ends and can cause misinterpretation of data (RT mispriming). This kind of artifact in RNA-seq based technologies is underappreciated and currently no adequate tools exist to computationally remove them from published data sets. We show that mispriming can occur with as little as two bases of complementarity at the 3' end of the primer followed by intermittent regions of complementarity. We also provide a computational pipeline that identifies cDNA reads produced from RT mispriming, allowing users to filter them out from any aligned data set. Using this analysis pipeline, we identify thousands of mispriming events in a dozen published data sets from diverse technologies including short RNA-seq, total/mRNA-seq, HITS-CLIP, and GRO-seq. We further show how RT mispriming can lead to misinterpretation of data. In addition to providing a solution to computationally remove RT-misprimed reads, we also propose an experimental solution to completely avoid RT-mispriming by performing RNA-seq using thermostable group II intron derived reverse transcriptase (TGIRT-seq).

Ago HITS-CLIP expands microRNA-mRNA interactions in nucleus and cytoplasm of gastric cancer cells.

BACKGROUND: Intensive investigations have identified a collection of microRNAs (miRNAs) and their functional machineries in cytoplasm. However, a comprehensive view of miRNAs and mRNAs in cytoplasm and nucleus has not been explored. This study aims to reveal the mechanisms of miRNA-RNA interactions in nucleus and cytoplasm. METHODS: In this study, the miRNAs and their target mRNAs in the Argonaute2 (Ago2) complex of nucleus and cytoplasm of gastric cancer cells were characterized using high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP). Then, the selected miRNAs were verified by Northern blot. The target mRNAs in the Argonaute2 (Ago2) complex of nucleus and cytoplasm of gastric cancer cells were analyzed through Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analysis. RESULTS: The results revealed that there were 243 miRNAs and 265 miRNAs in the Ago2 complexes of nucleus and cytoplasm, respectively. The majority of mature miRNAs existed in cytoplasm. The analysis of miRNA targetome from the Ago2 complexes indicated that a lot of mRNAs with high expression level existed in nucleus. The target genes of miRNAs in the Ago2 complexes of nucleus and cytoplasm played important roles in cell proliferation, cell differentiation, innate immune response and tumorigenesis. CONCLUSIONS: microRNA-mRNA interactions occur in nucleus and cytoplasm of gastric cancer cells. Therefore, our study demonstrated that miRNA-mRNA interactions not only took place in cytoplasm but also in nucleus.

Evaluation and control of miRNA-like off-target repression for RNA interference.

RNA interference (RNAi) has been widely adopted to repress specific gene expression and is easily achieved by designing small interfering RNAs (siRNAs) with perfect sequence complementarity to the intended target mRNAs. Although siRNAs direct Argonaute (Ago), a core component of the RNA-induced silencing complex (RISC), to recognize and silence target mRNAs, they also inevitably function as microRNAs (miRNAs) and suppress hundreds of off-targets. Such miRNA-like off-target repression is potentially detrimental, resulting in unwanted toxicity and phenotypes. Despite early recognition of the severity of miRNA-like off-target repression, this effect has often been overlooked because of difficulties in recognizing and avoiding off-targets. However, recent advances in genome-wide methods and knowledge of Ago-miRNA target interactions have set the stage for properly evaluating and controlling miRNA-like off-target repression. Here, we describe the intrinsic problems of miRNA-like off-target effects caused by canonical and noncanonical interactions. We particularly focus on various genome-wide approaches and chemical modifications for the evaluation and prevention of off-target repression to facilitate the use of RNAi with secured specificity.

An RNA Switch of a Large Exon of Ninein Is Regulated by the Neural Stem Cell Specific-RNA Binding Protein, Qki5.

A set of tissue-specific splicing factors are thought to govern alternative splicing events during neural progenitor cell (NPC)-to-neuron transition by regulating neuron-specific exons. Here, we propose one such factor, RNA-binding protein Quaking 5 (Qki5), which is specifically expressed in the early embryonic neural stem cells. We performed mRNA-SEQ (Sequence) analysis using mRNAs obtained by developing cerebral cortices in Qk (Quaking) conditional knockout (cKO) mice. As expected, we found a large number of alternative splicing changes between control and conditional knockouts relative to changes in transcript levels. DAVID (The Database for Annotation, Visualization and Integrated Discovery) and Metascape analyses suggested that the affected spliced genes are involved in axon development and microtubule-based processes. Among these, the mRNA coding for the Ninein protein is listed as one of Qki protein-dependent alternative splicing targets. Interestingly, this exon encodes a very long polypeptide (2121 nt), and has been previously defined as a dynamic RNA switch during the NPC-to-neuron transition. Additionally, we validated that the regulation of this large exon is consistent with the Qki5-dependent alternative exon inclusion mode suggested by our previous Qki5 HITS-CLIP (high throughput sequencing-cross linking immunoprecipitation) analysis. Taken together, these data suggest that Qki5 is an important factor for alternative splicing in the NPC-to-neuron transition.

Genome-wide identification of miR-200 targets reveals a regulatory network controlling cell invasion.

The microRNAs of the miR-200 family maintain the central characteristics of epithelia and inhibit tumor cell motility and invasiveness. Using the Ago-HITS-CLIP technology for transcriptome-wide identification of direct microRNA targets in living cells, along with extensive validation to verify the reliability of the approach, we have identified hundreds of miR-200a and miR-200b targets, providing insights into general features of miRNA target site selection. Gene ontology analysis revealed a predominant effect of miR-200 targets in widespread coordinate control of actin cytoskeleton dynamics. Functional characterization of the miR-200 targets indicates that they constitute subnetworks that underlie the ability of cancer cells to migrate and invade, including coordinate effects on Rho-ROCK signaling, invadopodia formation, MMP activity, and focal adhesions. Thus, the miR-200 family maintains the central characteristics of the epithelial phenotype by acting on numerous targets at multiple levels, encompassing both cytoskeletal effectors that control actin filament organization and dynamics, and upstream signals that locally regulate the cytoskeleton to maintain cell morphology and prevent cell migration.

CLIPSeqTools--a novel bioinformatics CLIP-seq analysis suite.

Immunoprecipitation of RNA binding proteins (RBPs) after in vivo crosslinking, coupled with sequencing of associated RNA footprints (HITS-CLIP, CLIP-seq), is a method of choice for the identification of RNA targets and binding sites for RBPs. Compared with RNA-seq, CLIP-seq analysis is widely diverse and depending on the RBPs that are analyzed, the approaches vary significantly, necessitating the development of flexible and efficient informatics tools. In this study, we present CLIPSeqTools, a novel, highly flexible computational suite that can perform analysis from raw sequencing data with minimal user input. It contains a wide array of tools to provide an in-depth view of CLIP-seq data sets. It supports extensive customization and promotes improvization, a critical virtue, since CLIP-seq analysis is rarely well defined a priori. To highlight CLIPSeqTools capabilities, we used the suite to analyze Ago-miRNA HITS-CLIP data sets that we prepared from human brains.

A novel mechanism of LIN-28 regulation of let-7 microRNA expression revealed by in vivo HITS-CLIP in C. elegans.

The evolutionarily conserved gene lin-28 encodes an RNA-binding protein and is an important regulator of the proper temporal succession of several developmental events in both invertebrates and vertebrates. At the cellular level, LIN-28 promotes stemness and proliferation, and inhibits differentiation, a feature best illustrated by its ability to induce pluripotency when ectopically expressed in human fibroblasts in combination with NANOG, OCT4, and SOX2. Mammalian LIN28 functions in part by regulating processing of the let-7 microRNA through a GGAG binding site in the pre-let-7's distal loop region. However, many human and animal let-7 precursors lack the GGAG binding motif. In order to dissect the molecular mechanisms underlying its biological functions in a living animal, we identified a map of LIN-28 interactions with the transcriptome by in vivo HITS-CLIP in Caenorhabditis elegans. LIN-28 binds a large pool of messenger RNAs, and a substantial fraction of the bona fide LIN-28 targets are involved in aspects of animal development. Furthermore, our data show that LIN-28 regulates the expression of the let-7 microRNA by binding its primary transcript in a previously unknown region, revealing a novel regulatory mechanism.

A multiprotein occupancy map of the mRNP on the 3' end of histone mRNAs.

The animal replication-dependent (RD) histone mRNAs are coordinately regulated with chromosome replication. The RD-histone mRNAs are the only known cellular mRNAs that are not polyadenylated. Instead, the mature transcripts end in a conserved stem-loop (SL) structure. This SL structure interacts with the stem-loop binding protein (SLBP), which is involved in all aspects of RD-histone mRNA metabolism. We used several genomic methods, including high-throughput sequencing of cross-linked immunoprecipitate (HITS-CLIP) to analyze the RNA-binding landscape of SLBP. SLBP was not bound to any RNAs other than histone mRNAs. We performed bioinformatic analyses of the HITS-CLIP data that included (i) clustering genes by sequencing read coverage using CVCA, (ii) mapping the bound RNA fragment termini, and (iii) mapping cross-linking induced mutation sites (CIMS) using CLIP-PyL software. These analyses allowed us to identify specific sites of molecular contact between SLBP and its RD-histone mRNA ligands. We performed in vitro crosslinking assays to refine the CIMS mapping and found that uracils one and three in the loop of the histone mRNA SL preferentially crosslink to SLBP, whereas uracil two in the loop preferentially crosslinks to a separate component, likely the 3'hExo. We also performed a secondary analysis of an iCLIP data set to map UPF1 occupancy across the RD-histone mRNAs and found that UPF1 is bound adjacent to the SLBP-binding site. Multiple proteins likely bind the 3' end of RD-histone mRNAs together with SLBP.

Improvements to the HITS-CLIP protocol eliminate widespread mispriming artifacts.

BACKGROUND: High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) allows for high resolution, genome-wide mapping of RNA-binding proteins. This methodology is frequently used to validate predicted targets of microRNA binding, as well as direct targets of other RNA-binding proteins. Hence, the accuracy and sensitivity of binding site identification is critical. RESULTS: We found that substantial mispriming during reverse transcription results in the overrepresentation of sequences complementary to the primer used for reverse transcription. Up to 45 % of peaks in publicly available HITS-CLIP libraries are attributable to this mispriming artifact, and the majority of libraries have detectable levels of mispriming. We also found that standard techniques for validating microRNA-target interactions fail to differentiate between artifactual peaks and physiologically relevant peaks. CONCLUSIONS: Here, we present a modification to the HITS-CLIP protocol that effectively eliminates this artifact and improves the sensitivity and complexity of resulting libraries.

Preferential binding of a stable G3BP ribonucleoprotein complex to intron-retaining transcripts in mouse brain and modulation of their expression in the cerebellum.

Neuronal granules play an important role in the localization and transport of translationally silenced messenger ribonucleoproteins in neurons. Among the factors associated with these granules, the RNA-binding protein G3BP1 (stress-granules assembly factor) is involved in neuronal plasticity and is induced in Alzheimer's disease. We immunopurified a stable complex containing G3BP1 from mouse brain and performed high-throughput sequencing and cross-linking immunoprecipitation to identify the associated RNAs. The G3BP-complex contained the deubiquitinating protease USP10, CtBP1 and the RNA-binding proteins Caprin-1, G3BP2a and splicing factor proline and glutamine rich, or PSF. The G3BP-complex binds preferentially to transcripts that retain introns, and to non-coding sequences like 3'-untranslated region and long non-coding RNAs. Specific transcripts with retained introns appear to be enriched in the cerebellum compared to the rest of the brain and G3BP1 depletion decreased this intron retention in the cerebellum of G3BP1 knockout mice. Among the enriched transcripts, we found an overrepresentation of genes involved in synaptic transmission, especially glutamate-related neuronal transmission. Notably, G3BP1 seems to repress the expression of the mature Grm5 (metabotropic glutamate receptor 5) transcript, by promoting the retention of an intron in the immature transcript in the cerebellum. Our results suggest that G3BP is involved in a new functional mechanism to regulate non-coding RNAs including intron-retaining transcripts, and thus have broad implications for neuronal gene regulation, where intron retention is widespread.

Experimental and Computational Considerations in the Study of RNA-Binding Protein-RNA Interactions.

After an RNA is transcribed, it undergoes a variety of processing steps that can change the encoded protein sequence (through alternative splicing and RNA editing), regulate the stability of the RNA, and control subcellular localization, timing, and rate of translation. The recent explosion in genomics techniques has enabled transcriptome-wide profiling of RNA processing in an unbiased manner. However, it has also brought with it both experimental challenges in developing improved methods to probe distinct processing steps, as well as computational challenges in data storage, processing, and analysis tools to enable large-scale interpretation in the genomics era. In this chapter we review experimental techniques and challenges in profiling various aspects of RNA processing, as well as recent efforts to develop analyses integrating multiple data sources and techniques to infer RNA regulatory networks.

Passenger strand loading in overexpression experiments using microRNA mimics.

MicroRNAs (miRNAs) are important regulators of gene function and manipulation of miRNAs is a central component of basic research. Modulation of gene expression by miRNA gain-of-function can be based on different approaches including transfection with miRNA mimics; artificial, chemically modified miRNA-like small RNAs. These molecules are intended to mimic the function of a miRNA guide strand while bypassing the maturation steps of endogenous miRNAs. Due to easy accessibility through commercial providers this approach has gained popularity, and accuracy is often assumed without prior independent testing. Our in silico analysis of over-represented sequence motifs in microarray expression data and sequencing of AGO-associated small RNAs indicate, however, that miRNA mimics may be associated with considerable side-effects due to the unwanted activity of the miRNA mimic complementary strand.

Global insights into alternative polyadenylation regulation.

Alternative pre-mRNA processing greatly increases the coding capacity of the human genome and regulatory factors involved in RNA processing play critical roles in tissue development and maintenance. Indeed, abnormal functions of RNA processing factors have been associated with a wide range of human diseases from cancer to neurodegenerative disorders. While many studies have emphasized the importance of alternative splicing (AS), recent high-throughput sequencing efforts have also allowed global surveys of alternative polyadenylation (APA). For the majority of pre-mRNAs, as well as some non-coding transcripts such as lncRNAs, APA selects different 3'-ends and thus modulates the availability of regulatory sites recognized by trans-acting regulatory effectors, including miRs and RNA binding proteins (RBPs). Here, we compare the available technologies for assessing global polyadenylation patterns, summarize the roles of auxiliary factors on APA, and discuss the impact of differential polyA site (pA) selection in the determination of cell fate, transformation and disease.