Project description:The induction of pluripotency or trans-differentiation of one cell type to another can be accomplished with cell lineage-specific transcription factors. Here we report that repression of a single RNA binding protein PTB, which occurs during normal brain development via the action of miR-124, is sufficient to induce trans-differentiation of fibroblasts into functional neurons. Besides its traditional role in regulated splicing, we show that PTB has a previously undocumented function in the regulation of microRNA functions, suppressing or enhancing microRNA targeting by competitive binding on target mRNA or altering local RNA secondary structure. A key event during neuronal induction is the relief of PTB-mediated blockage of microRNA action on multiple components of the REST complex, thereby de-repressing a large array of neuronal genes, including miR-124 and multiple neuronal-specific transcription factors, in non-neuronal cells. This converts a negative feedback loop to a positive one to elicit cellular reprogramming to the neuronal lineage. Examination of PTB regulated AGO2/microRNA targeting in Hela cells by CLIP-seq (two biological replicates) , paired-end RNA-seq (control and PTB knockdown) and 3’end stability RNA-seq (control and PTB knockdown)
Project description:Ultraviolet (UV) radiation is a major melanoma risk factor, yet underlying mechanisms remain poorly understood. Here we introduce a mouse model permitting fluorescence-aided melanocyte imaging and isolation following in vivo UV irradiation. We use expression profiling to show that activated neonatal skin melanocytes isolated following a melanomagenic UVB dose bear a distinct, persistent interferon-response signature, including genes associated with immunoevasion. UVB-induced melanocyte activation, characterized by aberrant growth and migration, was abolished by antibody-mediated systemic blockade of interferon-gamma (IFN-gamma), but not type-I interferons. IFN-gamma was produced by macrophages recruited to neonatal skin by UVB-induced chemokine receptor Ccr2 ligands. Admixed recruited skin macrophages enhanced transplanted melanoma growth by inhibiting apoptosis; notably, IFN-gamma blockade abolished macrophage-associated melanoma growth and survival. IFN-gamma-producing macrophages were identified in 70% of human melanomas examined. Our data reveal an unanticipated role for IFN-gamma in promoting melanocytic cell survival/immunoevasion, and suggest IFN-gamma-R signaling represents a novel therapeutic melanoma target. Biologic replicates of UVA- and UVB-treated mouse melanocytes, as well as untreated mouse melanocytes and mouse keratinocytes, were used to define melanocyte expression signatures associated with UV treatment.
Project description:To study protein-RNA interactions at single amino acid and single nucleotide resolution we developed a new approach, termed cross-linking of segmentally isotope labeled RNA and tandem mass spectrometry (CLIR-MS/MS). The method was developed using the PTBP1-EMCV IRES complex as a model system and additionally applied to the U1 snRNP complex. Data from both complexes are included in this submission.
Project description:MicroRNAs (miRNAs) are a large family of 19-22nt non-coding RNAs that post-transcriptionally regulate their mRNA targets. Computational algorithms predict that over half of all genes are regulated by miRNAs, yet approaches for experimental identification of miRNA binding sites are now emerging. To directly identify endogenous miRNA binding sites, we performed photo-crosslinking immunoprecipitation using antibodies against Ago2, followed by deep-sequencing of RNA tags (CLIP-seq) in mouse embryonic stem cells (mESCs). We also performed parallel CLIP-seq in Dicer null mESCs that lack mature miRNAs, allowing us to define whether the association of Ago2 with the identified sites was mediated by miRNAs. We include the exon-array expression data obtained from three sets of Dicer WT and Dicer Null mESCs.These data are used to determine genes that are differentially expressed between Dicer WT and Dicer Null conditions. Six samples (3 Dicer wild-type CLIP RNA libraries representing two biological replicates, 2 Dicer null CLIP RNA libraries, 1 short-RNA library from Dicer wild-type mESCs) were analyzed. Six total mESC samples were analyzed (3 Dicer WT, 3 Dicer Null). Expression values for probesets were summarized into a single per-gene value. The log fold change for Dicer_WT/Dicer_Null was defined as the difference between the mean expression in Dicer WT mESCs and the mean expression in the Dicer Null mESCs.
Project description:Background: In recent years, a variety of small RNAs derived from other RNAs with well-known functions such as tRNAs and snoRNAs, have been identified. The functional relevance of these RNAs is largely unknown. To gain insight into the complexity of snoRNA processing and the functional relevance of snoRNA-derived small RNAs, we sequenced long and short RNAs, small RNAs that co-precipitate with the Argonaute 2 protein and RNA fragments obtained in photoreactive nucleotide-enhanced crosslinking and immunoprecipitation (PAR-CLIP) of core snoRNA-associated proteins. Results: Analysis of these data sets revealed that many loci in the human genome reproducibly give rise to C/D box-like snoRNAs, whose expression and evolutionary conservation are typically less pronounced relative to the snoRNAs that are currently catalogued. We further found that virtually all C/D box snoRNAs are specifically processed inside the regions of terminal complementarity, retaining in the mature form only 4-5 nucleotides upstream of the C box and 2-5 nucleotides downstream of the D box. Sequencing of the total and Argonaute 2-associated populations of small RNAs revealed that despite their cellular abundance, C/D box-derived small RNAs are not efficiently incorporated into the Ago2 protein. Conclusions: We conclude that the human genome encodes a large number of snoRNAs that are processed along the canonical pathway and expressed at relatively low levels. Generation of snoRNA-derived processing products with alternative, particularly miRNA-like, functions appears to be uncommon. PAR-CLIP profiling for snoRNP core proteins NOP56, NOP58, Fibrillarin, and Dyskerin in HEK293 cells. Small RNA profiling using RNA-seq in HEK293 and HeLa cells, small RNA profiling using IP-seq of Ago2 associated small RNAs.
Project description:RNA-binding proteins (RBPs) determine RNA fate from synthesis to decay. Employing two complementary protocols for covalent UV crosslinking of RBPs to RNA, we describe a systematic, unbiased, and comprehensive approach, termed "interactome capture," to define the mRNA interactome of proliferating human HeLa cells. We identify 860 proteins that qualify as RBPs by biochemical and statistical criteria, adding more than 300 RBPs to those previously known and shedding light on RBPs in disease, RNA-binding enzymes of intermediary metabolism, RNA-binding kinases, and RNA-binding architectures. Unexpectedly, we find that many proteins of the HeLa mRNA interactome are highly intrinsically disordered and enriched in short repetitive amino acid motifs. Interactome capture is broadly applicable to study mRNA interactome composition and dynamics in varied biological settings.
Project description:FUS, an RNA binding protein was recently implicated in Amyotrophic Lateral Sclerosis (ALS). ALS is a fatal neurodegenerative disease. We report the identification of the conserved neuronal RNA targets of FUS and the assessment of the impact of FUS depletion on the neuronal transcriptome. We identified that FUS regulates splicing of conserved intron containing transcripts. FUS retains or excludes the conserved intron by binding to them. Identification of FUS neuronal targets using normal human brain samples and mouse neurons
Project description:Small RNA deep sequencing analysis was conducted on primary human fibroblasts infected with human cytomegalovirus (HCMV). HCMV-encoded miRNAs accumulated to ~20% of the total smRNA population at late stages of infection, and our analysis led to improvements in viral miRNA annotations and identification of novel HCMV miRNAs. Through crosslinking and immunoprecipitation of Argonaute-bound RNAs from infected cells, followed by high-throughput sequencing (Ago CLIP-seq), we obtained direct evidence for incorporation of all HCMV miRNAs into the endogenous host silencing machinery. Additionally, significant upregulation was observed during infection for a host miRNA cluster containing miR-96, miR-182 and miR-183. We also identified novel non-miRNA forms of virus-derived smRNAs, revealing greater complexity within the smRNA population during HCMV infection. High-throughput profiling of smRNAs, Ago1-, and Ago2-associated miRNAs from HCMV-infected fibroblast cells. Wild-type HCMV Towne (Genbank FJ616285.1) was used for these studies.
Project description:DELLA proteins act as hubs that relay environmental information to the multiple transcriptional circuits that control growth and development through physical interaction with transcription factors from different families. We have analyzed the presence of one DELLA protein at the Arabidopsis genome by chromatin immunoprecipitation coupled to large-scale sequencing and we find that it binds at the promoters of multiple genes. Enrichment analysis shows a strong preference for cis elements recognized by specific transcription factor families. In particular, we demonstrate that DELLA proteins are recruited by type-B ARABIDOPSIS RESPONSE REGULATORS (ARR) to the promoters of cytokinin-regulated genes, where they act as transcriptional co-activators. The biological relevance of this mechanism is underpinned by the necessity of simultaneous presence of DELLAs and ARRs to restrict root meristem growth and to promote photomorphogenesis. Provided are 3 biological replicates analysing RGA binding sites in Arabidopsis seedlings. ChIP-seq was performed on plants expressing RGA-GFP under the native RGA promoter and on non-transgenic control plants as reference
Project description:We developed a method for measuring non-specific background in PAR-CLIP data demonstrating that covalently crosslinked background binding is common, reproducible and apparently universal. Furthermore, we show that quantitative determination of background is essential for identifying targets of weakly binding RNA-binding proteins and can substantially improve motif analysis. To define background binding events in PAR-CLIP data we performed the standard PAR-CLIP protocol (Hafner et al., Cell 2010.) on lysates expressing a commonly used non-RBP control, FLAG-GFP. After FLAG-tag immunopurification of UV 365nm irradiated lysates prepared from cells supplemented with 4-thiouridine (4SU), RNA was partially digested with RNase T1, radiolabeled and separated by SDS-PAGE. Reads were sequenced by Illumina HiSeq. PAR-CLIP was also performed for HuR. Included as well is a total from lysates treated like PAR-CLIP, but without immunoprecipitation (see sample description for more detail).