Differential association of chromatin proteins identifies BAF60a/SMARCD1 as a regulator of embryonic stem cell differentiation.

Embryonic stem cells (ESCs) possess a distinct chromatin conformation maintained by specialized chromatin proteins. To identify chromatin regulators in ESCs, we developed a simple biochemical assay named D-CAP (differential chromatin-associated proteins), using brief micrococcal nuclease digestion of chromatin, followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Using D-CAP, we identified several differentially chromatin-associated proteins between undifferentiated and differentiated ESCs, including the chromatin remodeling protein SMARCD1. SMARCD1 depletion in ESCs led to altered chromatin and enhanced endodermal differentiation. Gene expression and chromatin immunoprecipitation sequencing (ChIP-seq) analyses suggested that SMARCD1 is both an activator and a repressor and is enriched at developmental regulators and that its chromatin binding coincides with H3K27me3. SMARCD1 knockdown caused H3K27me3 redistribution and increased H3K4me3 around the transcription start site (TSS). One of the identified SMARCD1 targets was Klf4. In SMARCD1-knockdown clones, KLF4, as well as H3K4me3 at the Klf4 locus, remained high and H3K27me3 was abolished. These results propose a role for SMARCD1 in restricting pluripotency and activating lineage pathways by regulating H3K27 methylation.

Blanks, a nuclear siRNA/dsRNA-binding complex component, is required for Drosophila spermiogenesis.

Small RNAs and a diverse array of protein partners control gene expression in eukaryotes through a variety of mechanisms. By combining siRNA affinity chromatography and mass spectrometry, we have identified the double-stranded RNA-binding domain protein Blanks to be an siRNA- and dsRNA-binding protein from Drosophila S2 cells. We find that Blanks is a nuclear factor that contributes to the efficiency of RNAi. Biochemical fractionation of a Blanks-containing complex shows that the Blanks complex is unlike previously described RNA-induced silencing complexes and associates with the DEAD-box helicase RM62, a protein previously implicated in RNA silencing. In flies, Blanks is highly expressed in testes tissues and is necessary for postmeiotic spermiogenesis, but loss of Blanks is not accompanied by detectable transposon derepression. Instead, genes related to innate immunity pathways are up-regulated in blanks mutant testes. These results reveal Blanks to be a unique component of a nuclear siRNA/dsRNA-binding complex that contributes to essential RNA silencing-related pathways in the male germ line.

Proteomics identification of Drosophila small interfering RNA-associated factors.

The Drosophila melanogaster RNA-induced silencing complex (RISC) forms a large ribonucleoprotein particle on small interfering RNAs (siRNAs) and catalyzes target mRNA cleavage during RNA interference (RNAi). Dicer-2, R2D2, Loquacious, and Argonaute-2 are examples of RISC-associated factors that are involved in RNAi. Holo-RISC is an approximately 80 S small interfering ribonucleoprotein, which suggests that there are many additional proteins that participate in the RNAi pathway. In this study, we used siRNA affinity capture combined with mass spectrometry to identify novel components of the Drosophila RNAi machinery. Our study identified both established RISC components and novel siRNA-associated factors, many of which contain domains that are consistent with potential roles in RNAi. Functional analysis of these novel siRNA-associated proteins suggests that these factors may play an important role in RNAi.

An inside job for siRNAs.

Among the three main categories of small silencing RNAs in insects and mammals-siRNAs, miRNAs, and piRNAs-siRNAs were thought to arise primarily from exogenous sources, whereas miRNAs and piRNAs arise from endogenous loci. Recent work in flies and mice reveals several classes of endogenous siRNAs (endo-siRNAs) that contribute to functions previously reserved for miRNAs and piRNAs, including gene regulation and transposon suppression.

The importance of RNA structure in RNA editing and a potential proofreading mechanism for correct guide RNA:pre-mRNA binary complex formation.

RNA editing in trypanosomes is a post-transcriptional process responsible for correcting the coding sequences of many mitochondrial mRNAs. Uridine bases are specifically added or deleted from mRNA by an enzymatic cascade in which a pre-edited mRNA is cleaved specifically, uridine bases are added or removed, and the corrected mRNA is ligated. The process is directed by RNA molecules, termed guide RNAs (gRNA). The ability of this class of small, non-coding RNA to function in RNA editing is essential for these organisms. Typically, gRNAs are transcribed independent of their cognate mRNA and anneal to form a binary RNA complex. An exception from this process is the cytochrome oxidase subunit II (COII) mRNA, which encodes its gRNA within its 3' untranslated region. This gRNA lacks the ability to function in trans. Using an in vitro editing assay, we find that improving thermodynamic stability to the anchor region through increased Watson-Crick base-pairing is sufficient to impart trans editing activity. We further show that a point mutation outside the known functional regions of a gRNA induces both a conformational rearrangement of the gRNA and causes a decrease in the rate of editing. Taken together, these results lead us to propose a model for a potential proofreading step in the formation of a gRNA:pre-edited mRNA binary complex. The mechanism relies on the thermodynamic stability supplied to the RNA complex through Watson-Crick base-pairing. Through mutations in the gRNA, we demonstrate the importance of gRNA structure to the RNA editing reaction.

The 3'-untranslated region of cytochrome oxidase II mRNA functions in RNA editing of African trypanosomes exclusively as a cis guide RNA.

RNA editing in trypanosomes is a post-transcriptional process responsible for correcting the coding sequences of many mitochondrial mRNAs. Uridines are specifically added or deleted from mRNA by an enzymatic cascade in which a pre-edited mRNA is specifically cleaved, uridines are added or removed, and the corrected mRNA is ligated. The process is directed by RNA molecules, termed guide RNAs (gRNA). The ability of this class of small, noncoding RNA to function in RNA editing is essential for these organisms. Typically, gRNAs are transcribed independent of the their cognate mRNA and anneal to form a binary RNA complex . An exception for this process may be cytochrome oxidase subunit II (COII) mRNA since a gene encoding a trans acting gRNA has not been identified. Using an in vitro editing assay we find that the 3' UTR of COII, indeed, functions as a guide for both the site and number of uridines added to the coding region of the COII mRNA. We further show that the guiding sequence within the COII 3' UTR can only function in COII editing when contiguous with the editing substrate, indicating that the 3' UTR of COII lacks sequence or structure information necessary to function as a trans-acting gRNA. While other RNAs have been shown to "guide" RNA processing reactions, our discovery that the COII 3' UTR directs editing of its cognate mRNA in cis, is a unique function for a 3' UTR. The findings described here have led us to propose a new model for the evolution of gRNAs in kinetoplastids.