High-throughput Sequencing Of RNA Isolated By Cross-linking Immunoprecipitation Research Articles

Genome-wide identification of microRNA targets reveals positive regulation of the Hippo pathway by miR-122 during liver development

Liver development is a highly complex process that is regulated by the orchestrated interplay of epigenetic regulators, transcription factors, and microRNAs (miRNAs). Owing to the lack of global in vivo targets of all miRNAs during liver development, the mechanisms underlying the dynamic control of hepatocyte differentiation by miRNAs remain elusive. Here, using Argonaute (Ago) high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) in the mouse liver at different developmental stages, we characterized massive Ago-binding RNAs and obtained a genome-wide map of liver miRNA-mRNA interactions. The dynamic changes of five clusters of miRNAs and their potential targets were identified to be differentially involved at specific stages, a dozen of high abundant miRNAs and their epigenetic regulation by super-enhancer were found during liver development. Remarkably, miR-122, a liver-specific and most abundant miRNA in newborn and adult livers, was found by its targetome and pathway reporter analyses to regulate the Hippo pathway, which is crucial for liver size control and homeostasis. Mechanistically, we further demonstrated that miR-122 negatively regulates the outcomes of the Hippo pathway transcription factor TEAD by directly targeting a number of hippo pathway regulators, including the coactivator TAZ and a key factor of the phosphatase complex PPP1CC, which contributes to the dephosphorylation of YAP, another coactivator downstream of the Hippo pathway. This study identifies for the first time the genome-wide miRNA targetomes during mouse liver development and demonstrates a novel mechanism of terminal differentiation of hepatocytes regulated by the miR-122/Hippo pathway in a coordinated manner. As the Hippo pathway plays important roles in cell proliferation and liver pathological processes like inflammation, fibrosis, and hepatocellular carcinoma (HCC), our study could also provide a new insight into the function of miR-122 in liver pathology.

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.

Genome-Wide Identification of MicroRNA Targets Reveals Positive Regulation of the Hippo Pathway by miR-122 During Liver Development

Liver development is a highly complex process that is regulated by the orchestrated interplay of epigenetic regulators, transcription factors and microRNAs (miRNAs). Owing to the lack of global in vivo targets of all miRNAs during liver development, the mechanisms underlying the dynamic control of hepatocyte differentiation by miRNAs remain elusive. Here, using Argonaute (Ago) high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) in the mouse liver at different developmental stages, we characterized massive Ago-binding RNAs and obtained a genome-wide map of liver miRNA-mRNA interactions. The dynamic changes of five clusters of miRNAs and their potential targets were identified to be differentially involved at specific stages, a dozen of high abundant miRNAs and their epigenetic regulation by super-enhancer were found during liver development. Remarkably, miR-122, a liver-specific and most abundant miRNA in newborn and adult livers, was found by its targetome and pathway reporter analyses to regulate the Hippo pathway, which is crucial for liver size control and homeostasis. Mechanistically, we further demonstrated that miR-122 negatively regulates the outcomes of the Hippo pathway transcription factor TEAD by directly targeting a number of hippo pathway regulators, including the coactivator Taz and a key factor of the phosphatase complex Ppp1cc, which contributes to the dephosphorylation of YAP, another coactivator downstream of the Hippo pathway. This study identifies for the first time the genome-wide miRNA targetome during mouse liver development and demonstrates a novel mechanism of terminal differentiation of hepatocytes regulated by the miR-122/Hippo pathway in a coordinated manner.

Identification of Hfq-binding RNAs in Caulobacter crescentus

ABSTRACTSmall RNAs are important for post-transcriptional regulation of gene expression, affecting stability and activity of their target mRNAs. The bacterial Sm-like protein Hfq is required to promote pairing between both RNAs when their sequence complementarity is limited. To provide a first global view on the post-transcriptional landscape of the α-proteobacterium Caulobacter crescentus, we have identified the Hfq-binding RNAs employing High-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP). A total of 261 RNAs, including 3 unannotated RNAs, were successfully identified and classified according to putative function. Moreover, possible interactions between the identified sRNAs with mRNA targets were postulated through computational target predictions.

RIG-I Detects Kaposi's Sarcoma-Associated Herpesvirus Transcripts in a RNA Polymerase III-Independent Manner.

ABSTRACTRetinoic acid-inducible gene I (RIG-I) is a cytosolic pathogen recognition receptor that initiates the innate immune response against many RNA viruses. We previously showed that RIG-I restricts Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation (J. A. West et al., J Virol 88:5778–5787, 2014, https://doi.org/10.1128/JVI.03226-13). In this study, we report that KSHV stimulates the RIG-I signaling pathway in a RNA polymerase (Pol) III-independent manner and subsequently induces type I interferon (IFN) responses. Knockdown or inhibition of RNA Pol III had no effect on beta interferon (IFN-β) induction by KSHV. By using high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP) approach, we identified multiple KSHV regions that give rise to RNA fragments binding to RIG-I, such as ORF810420-10496, Repeat region (LIR1)119059-119204, and ORF2543561-43650. The sequence dissimilarity between these fragments suggests that RIG-I detects a particular structure rather than a specific sequence motif. Synthesized ORF810420-10496 RNA stimulated RIG-I-dependent but RNA Pol III-independent IFN-β signaling. In summary, several KSHV RNAs are sensed by RIG-I in a RNA Pol III-independent manner.

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.

Cstf2t Regulates expression of histones and histone-like proteins in male germ cells.

Formation of the 3' ends of mature mRNAs requires recognition of the correct site within the last exon, cleavage of the nascent pre-mRNA, and, for most mRNAs, addition of a poly(A) tail. Several factors are involved in recognition of the correct 3'-end site. The cleavage stimulation factor (CstF) has three subunits, CstF-50 (gene symbol Cstf1), CstF-64 (Cstf2), and CstF-77 (Cstf3). Of these, CstF-64 is the RNA-binding subunit that interacts with the pre-mRNA downstream of the cleavage site. In male germ cells where CstF-64 is not expressed, a paralog, τCstF-64 (gene symbol Cstf2t) assumes its functions. Accordingly, Cstf2t knockout (Cstf2t-/- ) mice exhibit male infertility due to defective development of spermatocytes and spermatids. To discover differentially expressed genes responsive to τCstF-64, we performed RNA-Seq in seminiferous tubules from wild-type and Cstf2t-/- mice, and found that several histone and histone-like mRNAs were reduced in Cstf2t-/- mice. We further observed delayed accumulation of the testis-specific histone, H1fnt (formerly, H1t2 or Hanp1) in Cstf2t-/- mice. High-throughput sequence analysis of polyadenylation sites (A-seq) indicated reduced use of polyadenylation sites within a cluster downstream of H1fnt in knockout mice. However, high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP) was not consistent with a direct role of τCstF-64 in polyadenylation of H1fnt. These findings together suggest that the τCstF-64 may control other reproductive functions that are not directly linked to the formation of 3' ends of mature polyadenylated mRNAs during male germ cell formation.

Genome-wide analysis of influenza viral RNA and nucleoprotein association

Influenza A virus (IAV) genomes are composed of eight single-stranded RNA segments that are coated by viral nucleoprotein (NP) molecules. Classically, the interaction between NP and viral RNA (vRNA) is depicted as a uniform pattern of ‘beads on a string’. Using high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP), we identified the vRNA binding profiles of NP for two H1N1 IAV strains in virions. Contrary to the prevailing model for vRNA packaging, NP does not bind vRNA uniformly in the A/WSN/1933 and A/California/07/2009 strains, but instead each vRNA segment exhibits a unique binding profile, containing sites that are enriched or poor in NP association. Intriguingly, both H1N1 strains have similar yet distinct NP binding profiles despite extensive sequence conservation. Peaks identified by HITS-CLIP were verified as true NP binding sites based on insensitivity to DNA antisense oligonucleotide-mediated RNase H digestion. Moreover, nucleotide content analysis of NP peaks revealed that these sites are relatively G-rich and U-poor compared to the genome-wide nucleotide content, indicating an as-yet unidentified sequence bias for NP association in vivo. Taken together, our genome-wide study of NP–vRNA interaction has implications for the understanding of influenza vRNA architecture and genome packaging.

Argonaute proteins regulate HIV-1 multiply spliced RNA and viral production in a Dicer independent manner.

Argonaute (Ago) proteins associate with microRNAs (miRNAs) to form the core of the RNA-induced silencing complex (RISC) that mediates post-transcriptional gene silencing of target mRNAs. As key players in anti-viral defense, Ago proteins are thought to have the ability to interact with human immunodeficiency virus type 1 (HIV-1) RNA. However, the role of this interaction in regulating HIV-1 replication has been debated. Here, we used high throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP) to explore the interaction between Ago2 and HIV-1 RNA in infected cells. By only considering reads of 50 nucleotides length in our analysis, we identified more than 30 distinct binding sites for Ago2 along the viral RNA genome. Using reporter assays, we found four binding sites, located near splice donor sites, capable of repressing Luciferase gene expression in an Ago-dependent manner. Furthermore, inhibition of Ago1 and Ago2 levels in cells expressing HIV-1 led to an increase of viral multiply spliced transcripts and to a strong reduction in the extracellular CAp24 level. Depletion of Dicer did not affect these activities. Our results highlight a new role of Ago proteins in the control of multiply spliced HIV-1 transcript levels and viral production, independently of the miRNA pathway.

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. ResultsWe 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.ConclusionsHere, we present a modification to the HITS-CLIP protocol that effectively eliminates this artifact and improves the sensitivity and complexity of resulting libraries.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2675-5) contains supplementary material, which is available to authorized users.

Herpesvirus saimiri MicroRNAs Preferentially Target Host Cell Cycle Regulators

In latently infected marmoset T cells, Herpesvirus saimiri (HVS) expresses six microRNAs (known as miR-HSURs [H. saimiri U-rich RNAs]). The viral miR-HSURs are processed from chimeric primary transcripts, each containing a noncoding U-rich RNA (HSUR) and a pre-miRNA hairpin. To uncover the functions of miR-HSURs, we identified mRNA targets in infected cells using high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP). HITS-CLIP revealed hundreds of robust Argonaute (Ago) binding sites mediated by miR-HSURs that map to the host genome but few in the HVS genome. Gene ontology analysis showed that several pathways regulating the cell cycle are enriched among cellular targets of miR-HSURs. Interestingly, miR-HSUR4-3p represses expression of the p300 transcriptional coactivator by binding the open reading frame of its mRNA. miR-HSUR5-3p directly regulates BiP, an endoplasmic reticulum (ER)-localized chaperone facilitating maturation of major histocompatibility complex class I (MHC-I) and the antiviral response. miR-HSUR5-3p also robustly downregulates WEE1, a key negative regulator of cell cycle progression, leading to reduced phosphorylation of its substrate, cyclin-dependent kinase (Cdk1). Consistently, inhibition of miR-HSUR5-3p in HVS-infected cells decreases their proliferation. Together, our results shed light on the roles of viral miRNAs in cellular transformation and viral latency. Viruses express miRNAs during various stages of infection, suggesting that viral miRNAs play critical roles in the viral life cycle. Compared to protein-coding genes, the functions of viral miRNAs are not well understood. This is because it has been challenging to identify their mRNA targets. Here, we focused on the functions of the recently discovered HVS miRNAs, called miR-HSURs. HVS is an oncogenic gammaherpesvirus that causes acute T-cell lymphomas and leukemias in New World primates and transforms human T cells. A better understanding of HVS biology will help advance our knowledge of virus-induced oncogenesis. Because numerous cellular miRNAs play crucial roles in cancer, viral miRNAs from the highly oncogenic HVS might also be important for transformation. Here, we found that the miR-HSURs preferentially modulate expression of host cell cycle regulators, as well as antiviral response factors. Our work provides further insight into the functions of herpesviral miRNAs in virus-induced oncogenesis and latency.

HIV-1 RNAs are Not Part of the Argonaute 2 Associated RNA Interference Pathway in Macrophages.

BackgroundMiRNAs and other small noncoding RNAs (sncRNAs) are key players in post-transcriptional gene regulation. HIV-1 derived small noncoding RNAs (sncRNAs) have been described in HIV-1 infected cells, but their biological functions still remain to be elucidated. Here, we approached the question whether viral sncRNAs may play a role in the RNA interference (RNAi) pathway or whether viral mRNAs are targeted by cellular miRNAs in human monocyte derived macrophages (MDM).MethodsThe incorporation of viral sncRNAs and/or their target RNAs into RNA-induced silencing complex was investigated using photoactivatable ribonucleoside-induced cross-linking and immunoprecipitation (PAR-CLIP) as well as high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP), which capture Argonaute2-bound miRNAs and their target RNAs. HIV-1 infected monocyte-derived macrophages (MDM) were chosen as target cells, as they have previously been shown to express HIV-1 sncRNAs. In addition, we applied small RNA deep sequencing to study differential cellular miRNA expression in HIV-1 infected versus non-infected MDMs.Results and ConclusionPAR-CLIP and HITS-CLIP data demonstrated the absence of HIV-1 RNAs in Ago2-RISC, although the presence of a multitude of HIV-1 sncRNAs in HIV-1 infected MDMs was confirmed by small RNA sequencing. Small RNA sequencing revealed that 1.4% of all sncRNAs were of HIV-1 origin. However, neither HIV-1 derived sncRNAs nor putative HIV-1 target sequences incorporated into Ago2-RISC were identified suggesting that HIV-1 sncRNAs are not involved in the canonical RNAi pathway nor is HIV-1 targeted by this pathway in HIV-1 infected macrophages.

HITS-CLIP Analysis Uncovers a Link between the Kaposi’s Sarcoma-Associated Herpesvirus ORF57 Protein and Host Pre-mRNA Metabolism

The Kaposi’s sarcoma associated herpesvirus (KSHV) is an oncogenic virus that causes Kaposi’s sarcoma, primary effusion lymphoma (PEL), and some forms of multicentric Castleman’s disease. The KSHV ORF57 protein is a conserved posttranscriptional regulator of gene expression that is essential for virus replication. ORF57 is multifunctional, but most of its activities are directly linked to its ability to bind RNA. We globally identified virus and host RNAs bound by ORF57 during lytic reactivation in PEL cells using high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP). As expected, ORF57-bound RNA fragments mapped throughout the KSHV genome, including the known ORF57 ligand PAN RNA. In agreement with previously published ChIP results, we observed that ORF57 bound RNAs near the oriLyt regions of the genome. Examination of the host RNA fragments revealed that a subset of the ORF57-bound RNAs was derived from transcript 5´ ends. The position of these 5´-bound fragments correlated closely with the 5´-most exon-intron junction of the pre-mRNA. We selected four candidates (BTG1, EGR1, ZFP36, and TNFSF9) and analyzed their pre-mRNA and mRNA levels during lytic phase. Analysis of both steady-state and newly made RNAs revealed that these candidate ORF57-bound pre-mRNAs persisted for longer periods of time throughout infection than control RNAs, consistent with a role for ORF57 in pre-mRNA metabolism. In addition, exogenous expression of ORF57 was sufficient to increase the pre-mRNA levels and, in one case, the mRNA levels of the putative ORF57 targets. These results demonstrate that ORF57 interacts with specific host pre-mRNAs during lytic reactivation and alters their processing, likely by stabilizing pre-mRNAs. These data suggest that ORF57 is involved in modulating host gene expression in addition to KSHV gene expression during lytic reactivation.

Argonaute HITS-CLIP Reveals Global miRNA-mRNA Networks in Erythropoiesis

MicroRNAs (miRNAs) inhibit gene expression by recruiting the RNA-induced silencing complex (RISC) to specific sets of target mRNAs. However, it has been challenging to define precisely the miRNA-target mRNA interactions that occur within cells. One approach to rigorously characterizing these networks in cells of interest is to sequence mRNA fragments bound to the RISC component Argonaute using a method termed High-Throughput Sequencing of RNA isolated by Crosslinking Immunoprecipitation (HITS-CLIP). We used this approach to define miRNA-target mRNA interactions during erythropoiesis, a developmental process known to be regulated by miRNAs and dramatically impaired by loss of Argonaute function.We performed Argonaute HITS-CLIP on primary mouse fetal liver erythroblasts in 3 biological replicate experiments and used Bowtie tools to map the sequenced RNA fragments to the mouse genome. These sequences represent mature miRNAs and their bound RNA segments in erythroid cells. Remarkably, miR-451 accounted for 70% of the Ago-bound erythroid miRNA burden, with miR-142, miR-144, miR-21, miR-374 and miR-30 accounting for an additional 15%. The frequency of Ago-binding correlated well with total cellular miRNA abundance. To analyze miRNA-target interactions, we used Piranha tools to identify peaks of sequenced reads, focusing on those that map to protein coding mRNAs. We identified 3,670 peaks across the entire genome, most of which mapped to mRNA 3’ untranslated regions (UTRs) (45%) or protein coding regions (38%). The remaining peaks mapped to intergenic regions, introns, long noncoding RNA genes and pseudogenes.To identify miRNA-mRNA associations, we searched for base pair complementarity between HITS-CLIP peaks mapping to mRNAs and seed sequence matches to the top 20 miRNAs in erythroid cells, which account for 92% of total miRNAs. We matched 36% of all peaks to canonical seed sequences and 34% to non-canonical seed sequences of specific miRNAs. We compared miR451-target mRNA matches predicted by HITS-CLIP to mRNA expression datasets generated from wild-type (WT) and miR-144/451 knockout (KO) mouse fetal liver erythroblasts. mRNAs with canonical miR-451 peaks in 3’ UTRs were significantly upregulated in KO erythroblasts as compared to WT erythroblasts (p<0.0001), indicating that our HITS-CLIP study identifies true erythroid targets of miR-451. These include previously identified targets of miR-451 such as Cab39, Ywhaz and Vapa, further validating our study. We also identified previously unknown targets such as Copa and Reep5, which, along with Vapa, regulate vesicle trafficking in other cell systems, and Matr3, Patl1 and Ythdf2, which affect global mRNA stability. Study of these genes may provide further insight into the functions of miR-451 in erythropoiesis. In contrast, mRNAs with predicted miR-451 peaks within coding exons showed no change in expression. This indicates that not all Ago HITS-CLIP peaks reflect regulation of mRNA stability, and that the presence of 3’ UTR Ago peaks with canonical miRNA seed sequences predicts better this mode of post transcriptional control of gene expression. Analysis and comparison of mRNA translational regulation by 3’UTR and coding region-bound miRNAs is in progress.Overall, our results provide a comprehensive map of Ago-bound miRNAs and their biologically relevant target mRNAs in erythroid cells. This map will serve as a basis to better understand miRNA regulation of erythropoiesis. DisclosuresNo relevant conflicts of interest to declare.

Global effects of the DEAD‐box RNA helicase DeaD (CsdA) on gene expression over a broad range of temperatures

In Escherichia coli, activity of the global regulatory RNA binding protein CsrA is antagonized by two non-coding sRNAs, CsrB and CsrC, which sequester it away from its lower affinity mRNA targets. Transcription of csrB/C requires the BarA-UvrY two component signal transduction system, which responds to short chain carboxylates. We show that two DEAD-box RNA helicases, DeaD and SrmB, activate csrB/C expression by different pathways. DeaD facilitates uvrY translation by counteracting the inhibitory effect of long distance base-pairing between the uvrY mRNA leader and coding region, while SrmB does not affect UvrY or UvrY-phosphate levels. Contrary to the prevailing notion that these helicases act primarily at low temperatures, DeaD and SrmB activated csrB expression over a wide temperature range. High-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP) revealed in vivo interactions of DeaD with 39 mRNAs, including those of uvrY and 9 other regulatory genes. Studies on the expression of several of the identified genes revealed regulatory effects of DeaD in all cases and diverse temperature response patterns. Our findings uncover an expanded regulatory role for DeaD, which is mediated through novel mRNA targets, important global regulators and under physiological conditions that were considered to be incompatible with its function.

Msi2 Directly Regulates The TGF-β Signaling Pathway and Myeloid Lineage Bias In Hematopoietic Stem Cells

Hematopoiesis is a tightly regulated process in which different cell lineages of the blood and immune system are generated from hematopoietic stem cells (HSC). HSCs can self renew and also give rise to more differentiated progenitor cells through symmetric and asymmetric cell division respectively. Progenitors can further differentiate into more committed cells that can generate the mature lymphoid and myeloid compartments. In order to support a normal hematopoietic system HSCs must maintain normal cell fate decisions between symmetric and asymmetric divisions. Recent studies from our group and others have implicated Msi2 as a regulator of HSCs. Nevertheless, the exact role for Msi2 in HSCs and critical pathways regulated by Msi2 in these stem cells remains unclear. In addition to its high expression in normal HSC, MSI2 is upregulated in patients with poor clinical prognosis in acute myeloid leukemia and in the blast crisis phase of chronic myelogenous leukemia. To understand the role of Msi2 in normal HSCs, we are characterizing mice with a conditional deletion of Msi2 (Mx1-Cre::Msi2flox/flox mice). We previously demonstrated that the conditional ablation of Msi2 results in the failure of HSC maintenance due to a loss of quiescence and increased commitment divisions. Although Msi2 is critical for HSC engraftment, we observed a preferential requirement for Msi2 in the myeloid biased HSCs (My-HSCs). Based on the surface markers LSK CD34- CD150 high we found a ∼3 fold reduction in the frequency of My-HSCs and no differences in the frequency of the unbiased HSCs or in common lymphoid progenitors. Consistent with these results, we observed a dramatic decrease in chimerism in the myeloid versus lymphoid compartment of the Msi2 deleted cells in mice that were transplanted. In order to understand why hematopoietic stem and progenitors cells require Msi2, we previously performed global transcriptome profiling and RNA target analysis using high throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP). This overlap analysis implicated a variety of pathways including RNA translation, HSC self-renewal and TGF-β. The TGF-β pathway has been linked to the maintenance of both normal and leukemia stem cell self-renewal, and the control of myeloid biased HSCs. We examined the output of the TGF-β pathway in HSCs in the presence and absence of Msi2. We found reduced phosphorylation of Smad2/Smad3 in Msi2D/D HSCs grown in vitro or directly isolated from bone marrow. Recently, it has been reported that the loss of p57 in HSCs results in reduced quiescence and compensatory up-regulation of p27. Consistent with reduced signaling output of TGF-β, we observed a significant decrease in p57 expression and up-regulation of p27 in Msi2D/D LSKs. The effect of TGF-β treatment is biphasic in hematopoietic stem cells, with high levels of TGF-β blocking proliferation and low levels of this pathway activation leading to increased proliferation. We therefore examined the functional response of Msi2D/D HSCs to biphasic TGF-β signaling in vitro. Msi2D/D HSCs responded normally to high dose of exogenous TGF-β resulting in potent growth suppression. In contrast, when exposed to lower levels of TGF-β that has been shown to expand normal HSCs, Msi2D/D HSCs failed to respond, consistent with diminished sensitivity to the proliferative effects of TGF-β. These results demonstrate that the proliferative signals downstream of TGF-β signaling are impaired in the Msi2 deleted HSCs. Most intriguingly, our data suggests that Msi2 is required to maintain the response to TGF-β signaling and this contributes to the maintenance and fate of the My-HSCs. It will be interesting to find out if Msi2 is involved in the prevalence of My-HSCs during aging. Taken together, these results have important implications for understanding how the activity of RNA binding proteins contribute to HSC biology that govern normal tissue homeostasis. Disclosures: No relevant conflicts of interest to declare.

Cellular functions of the microprocessor

The microprocessor is a complex comprising the RNase III enzyme Drosha and the double-stranded RNA-binding protein DGCR8 (DiGeorge syndrome critical region 8 gene) that catalyses the nuclear step of miRNA (microRNA) biogenesis. DGCR8 recognizes the RNA substrate, whereas Drosha functions as an endonuclease. Recent global analyses of microprocessor and Dicer proteins have suggested novel functions for these components independent of their role in miRNA biogenesis. A HITS-CLIP (high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation) experiment designed to identify novel substrates of the microprocessor revealed that this complex binds and regulates a large variety of cellular RNAs. The microprocessor-mediated cleavage of several classes of RNAs not only regulates transcript levels, but also modulates alternative splicing events, independently of miRNA function. Importantly, DGCR8 can also associate with other nucleases, suggesting the existence of alternative DGCR8 complexes that may regulate the fate of a subset of cellular RNAs. The aim of the present review is to provide an overview of the diverse functional roles of the microprocessor.

MicroRNAs Control Intestinal Epithelial Differentiation, Architecture, and Barrier Function

Whereas the importance of microRNA (miRNA) for the development of several tissues is well established, its role in the intestine is unknown. We aimed to quantify the complete miRNA expression profile of the mammalian intestinal mucosa and to determine the contribution of miRNAs to intestinal homeostasis using genetic means. We determined the miRNA transcriptome of the mouse intestinal mucosa using ultrahigh throughput sequencing. Using high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP), we identified miRNA-messenger RNA target relationships in the jejunum. We employed gene ablation of the obligatory miRNA-processing enzyme Dicer1 to derive mice deficient for all miRNAs in intestinal epithelia. miRNA abundance varies dramatically in the intestinal mucosa, from 1 read per million to 250,000. Of the 453 miRNA families identified, mmu-miR-192 is the most highly expressed in both the small and large intestinal mucosa, and there is a 53% overlap in the top 15 expressed miRNAs between the 2 tissues. The intestinal epithelium of Dicer1(loxP/loxP);Villin-Cre mutant mice is disorganized, with a decrease in goblet cells, a dramatic increase in apoptosis in crypts of both jejunum and colon, and accelerated jejunal cell migration. Furthermore, intestinal barrier function is impaired in Dicer1-deficient mice, resulting in intestinal inflammation with lymphocyte and neutrophil infiltration. Our list of miRNA-messenger RNA targeting relationships in the small intestinal mucosa provides insight into the molecular mechanisms behind the phenotype of Dicer1 mutant mice. We have identified all intestinal miRNAs and shown using gene ablation of Dicer1 that miRNAs play a vital role in the differentiation and function of the intestinal epithelium.

Aiding in the evolution of translational medicine: “Bridging Academia and Industry” in the field of neuroscience

Aiding in the evolution of translational medicine: “Bridging Academia and Industry” in the field of neuroscience

HITS-CLIP yields genome-wide insights into brain alternative RNA processing

SummaryProtein-RNA interactions play critical roles in all aspects of gene expression. Here we develop a genome-wide means of mapping protein-RNA binding sites in vivo, by high throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP). HITS-CLIP analysis of the neuron-specific splicing factor Nova2 revealed extremely reproducible RNA binding maps in multiple mouse brains. These maps provide genome-wide in vivo biochemical footprints confirming the previous prediction that the position of Nova binding determines the outcome of alternative splicing; moreover, they are sufficiently powerful to predict Nova action de novo. HITS-CLIP revealed a large number of Nova-RNA interactions in 3′ UTRs, leading to the discovery that Nova regulates alternative polyadenylation in the brain. HITS-CLIP, therefore, provides a robust, unbiased means to identify functional protein-RNA interactions in vivo.