LIN28 coordinately promotes nucleolar/ribosomal functions and represses the 2C-like transcriptional program in pluripotent stem cells.

LIN28 is an RNA binding protein with important roles in early embryo development, stem cell differentiation/reprogramming, tumorigenesis and metabolism. Previous studies have focused mainly on its role in the cytosol where it interacts with Let-7 microRNA precursors or mRNAs, and few have addressed LIN28's role within the nucleus. Here, we show that LIN28 displays dynamic temporal and spatial expression during murine embryo development. Maternal LIN28 expression drops upon exit from the 2-cell stage, and zygotic LIN28 protein is induced at the forming nucleolus during 4-cell to blastocyst stage development, to become dominantly expressed in the cytosol after implantation. In cultured pluripotent stem cells (PSCs), loss of LIN28 led to nucleolar stress and activation of a 2-cell/4-cell-like transcriptional program characterized by the expression of endogenous retrovirus genes. Mechanistically, LIN28 binds to small nucleolar RNAs and rRNA to maintain nucleolar integrity, and its loss leads to nucleolar phase separation defects, ribosomal stress and activation of P53 which in turn binds to and activates 2C transcription factor Dux. LIN28 also resides in a complex containing the nucleolar factor Nucleolin (NCL) and the transcriptional repressor TRIM28, and LIN28 loss leads to reduced occupancy of the NCL/TRIM28 complex on the Dux and rDNA loci, and thus de-repressed Dux and reduced rRNA expression. Lin28 knockout cells with nucleolar stress are more likely to assume a slowly cycling, translationally inert and anabolically inactive state, which is a part of previously unappreciated 2C-like transcriptional program. These findings elucidate novel roles for nucleolar LIN28 in PSCs, and a new mechanism linking 2C program and nucleolar functions in PSCs and early embryo development.

Phase separation of RNA-binding protein promotes polymerase binding and transcription.

An RNA-involved phase-separation model has been proposed for transcription control. However, the molecular links that connect RNA to the transcription machinery remain missing. Here we find that RNA-binding proteins (RBPs) constitute half of the chromatin proteome in embryonic stem cells (ESCs), some being colocalized with RNA polymerase (Pol) II at promoters and enhancers. Biochemical analyses of representative RBPs show that the paraspeckle protein PSPC1 inhibits the RNA-induced premature release of Pol II, and makes use of RNA as multivalent molecules to enhance the formation of transcription condensates and subsequent phosphorylation and release of Pol II. This synergistic interplay enhances polymerase engagement and activity via the RNA-binding and phase-separation activities of PSPC1. In ESCs, auxin-induced acute degradation of PSPC1 leads to genome-wide defects in Pol II binding and nascent transcription. We propose that promoter-associated RNAs and their binding proteins synergize the phase separation of polymerase condensates to promote active transcription.

Homotypic clustering of L1 and B1/Alu repeats compartmentalizes the 3D genome.

Organization of the genome into euchromatin and heterochromatin appears to be evolutionarily conserved and relatively stable during lineage differentiation. In an effort to unravel the basic principle underlying genome folding, here we focus on the genome itself and report a fundamental role for L1 (LINE1 or LINE-1) and B1/Alu retrotransposons, the most abundant subclasses of repetitive sequences, in chromatin compartmentalization. We find that homotypic clustering of L1 and B1/Alu demarcates the genome into grossly exclusive domains, and characterizes and predicts Hi-C compartments. Spatial segregation of L1-rich sequences in the nuclear and nucleolar peripheries and B1/Alu-rich sequences in the nuclear interior is conserved in mouse and human cells and occurs dynamically during the cell cycle. In addition, de novo establishment of L1 and B1 nuclear segregation is coincident with the formation of higher-order chromatin structures during early embryogenesis and appears to be critically regulated by L1 and B1 transcripts. Importantly, depletion of L1 transcripts in embryonic stem cells drastically weakens homotypic repeat contacts and compartmental strength, and disrupts the nuclear segregation of L1- or B1-rich chromosomal sequences at genome-wide and individual sites. Mechanistically, nuclear co-localization and liquid droplet formation of L1 repeat DNA and RNA with heterochromatin protein HP1α suggest a phase-separation mechanism by which L1 promotes heterochromatin compartmentalization. Taken together, we propose a genetically encoded model in which L1 and B1/Alu repeats blueprint chromatin macrostructure. Our model explains the robustness of genome folding into a common conserved core, on which dynamic gene regulation is overlaid across cells.

Transcriptome-Wide Mapping of Protein-RNA Interactions.

RNA and RNA-binding proteins (RBPs) control multiple biological processes. The spatial and temporal arrangement of RNAs and RBPs underlies the delicate regulation of these processes. The strategy called CLIP-seq (cross-linking and immunoprecipitation) has been developed to capture endogenous protein-RNA interactions with UV cross-linking followed by immunoprecipitation. Despite the wide use of conventional CLIP-seq method in RBP study, the CLIP experiment is limited by the availability of the high-quality antibodies, potential contaminants from the co-purified RBPs, requirement of isotope manipulation, and potential loss of information during tedious experimental procedure. Here we described a modified CLIP-seq method called FbioCLIP-seq using the FLAG-Biotin tag tandem purification. Through tandem purification and stringent wash condition, almost all the interacting RNA-binding proteins are removed; thus the indirect interacting RNAs mediated by these co-purified RBPs are also decreased. Our FbioCLIP-seq method allows efficient detection of direct protein-bound RNAs without SDS-PAGE and membrane transfer procedure in an isotope-free and protein-specific antibody-free manner.

Transcriptome-Wide Profiling of Protein-RNA Interactions by Cross-Linking and Immunoprecipitation Mediated by FLAG-Biotin Tandem Purification.

RNA and RNA-binding proteins (RBPs) control multiple biological processes. The spatial and temporal arrangement of RNAs and RBPs underlies the delicate regulation of these processes. A strategy called CLIP-seq (cross-linking and immunoprecipitation) has been developed to capture endogenous protein-RNA interactions with UV cross-linking followed by immunoprecipitation. Despite the wide use of conventional CLIP-seq method in RBP study, the CLIP method is limited by the availability of high-quality antibodies, potential contaminants from the copurified RBPs, requirement of isotope manipulation, and potential loss of information during a tedious experimental procedure. Here we describe a modified CLIP-seq method called FbioCLIP-seq using the FLAG-biotin tag tandem purification. Through tandem purification and stringent wash conditions, almost all the interacting RNA-binding proteins are removed. Thus, the RNAs interacting indirectly mediated by these copurified RBPs are also reduced. Our FbioCLIP-seq method allows efficient detection of direct protein-bound RNAs without SDS-PAGE and membrane transfer procedures in an isotope-free and protein-specific antibody-free manner.

RNA Targets Ribogenesis Factor WDR43 to Chromatin for Transcription and Pluripotency Control.

Transcription regulation underlies stem cell function and development. Here, we elucidate an unexpected role of an essential ribogenesis factor, WDR43, as a chromatin-associated RNA-binding protein (RBP) and release factor in modulating the polymerase (Pol) II activity for pluripotency regulation. WDR43 binds prominently to promoter-associated noncoding/nascent RNAs, occupies thousands of gene promoters and enhancers, and interacts with the Pol II machinery in embryonic stem cells (ESCs). Nascent transcripts and transcription recruit WDR43 to active promoters, where WDR43 facilitates releases of the elongation factor P-TEFb and paused Pol II. Knockdown of WDR43 causes genome-wide defects in Pol II release and pluripotency-associated gene expression. Importantly, auxin-mediated rapid degradation of WDR43 drastically reduces Pol II activity, precluding indirect consequences. These results reveal an RNA-mediated recruitment and feedforward regulation on transcription and demonstrate an unforeseen role of an RBP in promoting Pol II elongation and coordinating high-level transcription and translation in ESC pluripotency.

Insight into novel RNA-binding activities via large-scale analysis of lncRNA-bound proteome and IDH1-bound transcriptome.

RNA-binding proteins (RBPs) play pivotal roles in directing RNA fate and function. Yet the current annotation of RBPs is largely limited to proteins carrying known RNA-binding domains. To systematically reveal dynamic RNA-protein interactions, we surveyed the human proteome by a protein array-based approach and identified 671 proteins with RNA-binding activity. Among these proteins, 525 lack annotated RNA-binding domains and are enriched in transcriptional and epigenetic regulators, metabolic enzymes, and small GTPases. Using an improved CLIP (crosslinking and immunoprecipitation) method, we performed genome-wide target profiling of isocitrate dehydrogenase 1 (IDH1), a novel RBP. IDH1 binds to thousands of RNA transcripts with enriched functions in transcription and chromatin regulation, cell cycle and RNA processing. Purified IDH1, but not an oncogenic mutant, binds directly to GA- or AU-rich RNA that are also enriched in IDH1 CLIP targets. Our study provides useful resources of unconventional RBPs and IDH1-bound transcriptome, and convincingly illustrates, for the first time, the in vivo and in vitro RNA targets and binding preferences of IDH1, revealing an unanticipated complexity of RNA regulation in diverse cellular processes.

RNA-dependent chromatin targeting of TET2 for endogenous retrovirus control in pluripotent stem cells.

Ten-eleven translocation (TET) proteins play key roles in the regulation of DNA-methylation status by oxidizing 5-methylcytosine (5mC) to generate 5-hydroxymethylcytosine (5hmC), which can both serve as a stable epigenetic mark and participate in active demethylation. Unlike the other members of the TET family, TET2 does not contain a DNA-binding domain, and it remains unclear how it is recruited to chromatin. Here we show that TET2 is recruited by the RNA-binding protein Paraspeckle component 1 (PSPC1) through transcriptionally active loci, including endogenous retroviruses (ERVs) whose long terminal repeats (LTRs) have been co-opted by mammalian genomes as stage- and tissue-specific transcriptional regulatory modules. We found that PSPC1 and TET2 contribute to ERVL and ERVL-associated gene regulation by both transcriptional repression via histone deacetylases and post-transcriptional destabilization of RNAs through 5hmC modification. Our findings provide evidence for a functional role of transcriptionally active ERVs as specific docking sites for RNA epigenetic modulation and gene regulation.