Identification of miR-128 Target mRNAs That Are Expressed in B Cells Using a Modified Dual Luciferase Vector.

MicroRNAs (miRNAs) are 21-25 nucleotide long non-coding ribonucleic acids that modulate gene expression by degrading transcripts or inhibiting translation. The miRNA miR-128, originally thought to be brain-specific, was later also found in immune cells. To identify a valuable immune cell model system to modulate endogenous miR-128 amounts and to validate predicted miR-128 target mRNAs in B cells, we first investigated miR-128 expression using Northern blot analysis in several cell lines representing different stages of B cell development. The results showed that only primary brain cells showed significant levels of mature miR-128. To study the function of miR-128 in immune cells, we modified dual luciferase vectors to allow easy transfer of 3' UTR fragments with predicted miR-128 binding sites from widely used single to dual luciferase vectors. Comparison of in silico predicted miR-128-regulated mRNAs in single and dual luciferase constructs yielded similar results, validating the dual luciferase vector for miRNA target analysis. Furthermore, we confirmed miR-128-regulated mRNAs identified in silico and in vivo using the Ago HITS-CLIP technique and known to be expressed in B cells using the dual luciferase assay. In conclusion, this study provides new insights into the expression and function of miR-128 by validating novel target mRNAs expressed in B cells and identifying additional pathways likely controlled by this miRNA in the immune system.

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.

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.

Solid-Support Directional (SSD) RNA-Seq as a Companion Method to CLIP-Seq.

CLIP-Seq (Deep Sequencing after in vivo Crosslinking and Immunoprecipitation, HITS-CLIP) has emerged as a key method for the study of RNA-binding proteins (RBPs), as it can scrutinize the RNAs bound by an RBP in vivo, with minimum manipulation of biological samples. CLIP-Seq is best used to reveal changes of the RNA cargo of an RBP and differences on binding patterns of the bound RNAs in living cells in different genetic backgrounds or after experimental treatment, rather than simply identifying RNA species. It is therefore crucial that a reference of the steady state levels of the RNAs present in the samples used for the CLIP-Seq experiment is included in the bioinformatic analysis. A simple directional RNA-Seq method was developed that uses the same oligonucleotides and the same PCR amplification steps as our CLIP-Seq method, which therefore can be analyzed using the same bioinformatic pipeline as the CLIP-Seq data. This greatly simplifies and streamlines the analysis process, and at the same time reduces the chances of protocol-specific artifacts and biases interfering with data interpretation. Some considerations on ways to integrate CLIP-Seq and RNA-Seq analyses are also provided herein.

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.

Functional microRNA targetome undergoes degeneration-induced shift in the retina.

BACKGROUND: MicroRNA (miRNA) play a significant role in the pathogenesis of complex neurodegenerative diseases including age-related macular degeneration (AMD), acting as post-transcriptional gene suppressors through their association with argonaute 2 (AGO2) - a key member of the RNA Induced Silencing Complex (RISC). Identifying the retinal miRNA/mRNA interactions in health and disease will provide important insight into the key pathways miRNA regulate in disease pathogenesis and may lead to potential therapeutic targets to mediate retinal degeneration. METHODS: To identify the active miRnome targetome interactions in the healthy and degenerating retina, AGO2 HITS-CLIP was performed using a rodent model of photoreceptor degeneration. Analysis of publicly available single-cell RNA sequencing (scRNAseq) data was performed to identify the cellular location of AGO2 and key members of the microRNA targetome in the retina. AGO2 findings were verified by in situ hybridization (RNA) and immunohistochemistry (protein). RESULTS: Analysis revealed a similar miRnome between healthy and damaged retinas, however, a shift in the active targetome was observed with an enrichment of miRNA involvement in inflammatory pathways. This shift was further demonstrated by a change in the seed binding regions of miR-124-3p, the most abundant retinal AGO2-bound miRNA, and has known roles in regulating retinal inflammation. Additionally, photoreceptor cluster miR-183/96/182 were all among the most highly abundant miRNA bound to AGO2. Following damage, AGO2 expression was localized to the inner retinal layers and more in the OLM than in healthy retinas, indicating a locational miRNA response to retinal damage. CONCLUSIONS: This study provides important insight into the alteration of miRNA regulatory activity that occurs as a response to retinal degeneration and explores the miRNA-mRNA targetome as a consequence of retinal degenerations. Further characterisation of these miRNA/mRNA interactions in the context of the degenerating retina may provide an important insight into the active role these miRNA may play in diseases such as AMD.

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.

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.

Argonaute-CLIP delineates versatile, functional RNAi networks in Aedes aegypti, a major vector of human viruses.

Argonaute (AGO) proteins bind small RNAs to silence complementary RNA transcripts, and they are central to RNA interference (RNAi). RNAi is critical for regulation of gene expression and antiviral defense in Aedes aegypti mosquitoes, which transmit Zika, chikungunya, dengue, and yellow fever viruses. In mosquitoes, AGO1 mediates miRNA interactions, while AGO2 mediates siRNA interactions. We applied AGO-crosslinking immunoprecipitation (AGO-CLIP) for both AGO1 and AGO2, and we developed a universal software package for CLIP analysis (CLIPflexR), identifying 230 small RNAs and 5,447 small RNA targets that comprise a comprehensive RNAi network map in mosquitoes. RNAi network maps predicted expression levels of small RNA targets in specific tissues. Additionally, this resource identified unexpected, context-dependent AGO2 target preferences, including endogenous viral elements and 3'UTRs. Finally, contrary to current thinking, mosquito AGO2 repressed imperfect targets. These findings expand our understanding of small RNA networks and have broad implications for the study of antiviral RNAi.

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.

miR-15/16 Restrain Memory T Cell Differentiation, Cell Cycle, and Survival.

Coordinate control of T cell proliferation, survival, and differentiation are essential for host protection from pathogens and cancer. Long-lived memory cells, whose precursors are formed during the initial immunological insult, provide protection from future encounters, and their generation is the goal of many vaccination strategies. microRNAs (miRNAs) are key nodes in regulatory networks that shape effective T cell responses through the fine-tuning of thousands of genes. Here, using compound conditional mutant mice to eliminate miR-15/16 family miRNAs in T cells, we show that miR-15/16 restrict T cell cycle, survival, and memory T cell differentiation. High throughput sequencing of RNA isolated by cross-linking immunoprecipitation of AGO2 combined with gene expression analysis in miR-15/16-deficient T cells indicates that these effects are mediated through the direct inhibition of an extensive network of target genes within pathways critical to cell cycle, survival, and memory.

Investigating miRNA-lncRNA Interactions: Computational Tools and Resources.

In the last two decades noncoding RNAs have been the recipients of increasing scientific interest. In particular, miRNAs, short (~22 nts) noncoding transcripts, have been thoroughly investigated since their essential role in posttranscriptional gene expression regulation had been established in the early 2000s. With the advent and the advancements of high-throughput sequencing technologies in recent years, long noncoding RNAs have also started to emerge as important actors in cellular functions and processes. Such transcripts, on average longer than 200 nt, whose functions have yet to be fully characterized, have recently been identified as regulatory elements of the RNAi pathway, harboring several miRNA response elements, uncovering the phenomena of competing endogenous RNAs (ceRNAs), or "sponge RNAs." The present chapter aims to provide a brief update on the actual biomedical relevance of ceRNAs, together with a summary of resources, tools, and practical examples of their application to aid researchers in the discovery and further elucidation of lncRNA-miRNA interactions.

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.

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.

HITS-CLIP reveals sex-differential RNA binding and alterative splicing regulation of SRm160 in Drosophila.

Serine/arginine (SR)-rich proteins are critical for the regulation of alternative splicing (AS), which generates multiple mRNA isoforms from one gene and provides protein diversity for cell differentiation and tissue development. Genetic evidence suggests that Drosophila genital-specific overexpression of SR-related nuclear matrix protein of 160 kDa (SRm160), an SR protein with a PWI RNA-binding motif, causes defective development only in male flies and results in abnormal male genital structures and abnormal testis. However, the molecular characterization of SRm160 is limited. Using the high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) method in two sex-specific embryonic cell lines, S2 from the male and Kc from the female, we first identified the genome-wide RNA-binding characteristics of SRm160, which preferred binding to the exonic tri-nucleotide repeats GCA and AAC. We then validated this binding through both in vitro gel-shift assay and in vivo splicing of minigenes and found that SRm160 level affects AS of many transcripts. Furthermore, we identified 492 differential binding sites (DBS) of SRm160 varying between the two sex-specific cell lines. Among these DBS-containing genes, splicing factors were highly enriched, including transformer, a key regulator in the sex determination cascade. Analyses of fly mutants demonstrated that the SRm160 level affects AS isoforms of transformer. These findings shed crucial light on SRm160's RNA-binding specificity and regulation of AS in Drosophila sex determination and development.

Contemporary Ribonomics Methods for Viral microRNA Target Analysis.

Numerous cellular processes are regulated by microRNAs (miRNAs), both cellular and viral. Elucidating the targets of miRNAs has become an active area of research. An important method in this field is cross-linking and immunoprecipitation (CLIP), where cultured cells or tissues are UV-irradiated to cross-link protein and nucleic acid, the RNA binding protein of interest is immunoprecipitated, and the RNAs pulled down with the protein are isolated, reverse-transcribed, and analyzed by sequencing. CLIP using antibody against Argonaute (Ago), which binds to both miRNA and mRNA as they interact in RISC, has allowed researchers to uncover a large number of miRNA targets. Coupled with high-throughput sequencing, CLIP has been useful for revealing miRNA targetomes for the γ-herpesviruses Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Variants on the CLIP protocol are described, with the benefits and drawbacks of each. In particular, the most recent methods involving RNA⁻RNA ligation to join the miRNA and its RNA target have aided in target identification. Lastly, data supporting biologically meaningful interactions between miRNAs and long non-coding RNAs (lncRNAs) are reviewed. In summary, ribonomics-based miRNA targetome analysis has expanded our understanding of miRNA targeting and has provided a rich resource for EBV and KSHV research with respect to pathogenesis and tumorigenesis.

omniCLIP: probabilistic identification of protein-RNA interactions from CLIP-seq data.

CLIP-seq methods allow the generation of genome-wide maps of RNA binding protein - RNA interaction sites. However, due to differences between different CLIP-seq assays, existing computational approaches to analyze the data can only be applied to a subset of assays. Here, we present a probabilistic model called omniCLIP that can detect regulatory elements in RNAs from data of all CLIP-seq assays. omniCLIP jointly models data across replicates and can integrate background information. Therefore, omniCLIP greatly simplifies the data analysis, increases the reliability of results and paves the way for integrative studies based on data from different assays.

Non-Uniform and Non-Random Binding of Nucleoprotein to Influenza A and B Viral RNA.

The genomes of influenza A and B viruses have eight, single-stranded RNA segments that exist in the form of a viral ribonucleoprotein complex in association with nucleoprotein (NP) and an RNA-dependent RNA polymerase complex. We previously used high-throughput RNA sequencing coupled with crosslinking immunoprecipitation (HITS-CLIP) to examine where NP binds to the viral RNA (vRNA) and demonstrated for two H1N1 strains that NP binds vRNA in a non-uniform, non-random manner. In this study, we expand on those initial observations and describe the NP-vRNA binding profile for a seasonal H3N2 and influenza B virus. We show that, similar to H1N1 strains, NP binds vRNA in a non-uniform and non-random manner. Each viral gene segment has a unique NP binding profile with areas that are enriched for NP association as well as free of NP-binding. Interestingly, NP-vRNA binding profiles have some conservation between influenza A viruses, H1N1 and H3N2, but no correlation was observed between influenza A and B viruses. Our study demonstrates the conserved nature of non-uniform NP binding within influenza viruses. Mapping of the NP-bound vRNA segments provides information on the flexible NP regions that may be involved in facilitating assembly.

ZFP36 RNA-binding proteins restrain T cell activation and anti-viral immunity.

Dynamic post-transcriptional control of RNA expression by RNA-binding proteins (RBPs) is critical during immune response. ZFP36 RBPs are prominent inflammatory regulators linked to autoimmunity and cancer, but functions in adaptive immunity are less clear. We used HITS-CLIP to define ZFP36 targets in mouse T cells, revealing unanticipated actions in regulating T-cell activation, proliferation, and effector functions. Transcriptome and ribosome profiling showed that ZFP36 represses mRNA target abundance and translation, notably through novel AU-rich sites in coding sequence. Functional studies revealed that ZFP36 regulates early T-cell activation kinetics cell autonomously, by attenuating activation marker expression, limiting T cell expansion, and promoting apoptosis. Strikingly, loss of ZFP36 in vivo accelerated T cell responses to acute viral infection and enhanced anti-viral immunity. These findings uncover a critical role for ZFP36 RBPs in restraining T cell expansion and effector functions, and suggest ZFP36 inhibition as a strategy to enhance immune-based therapies.

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).