MicroRNAs (miRNAs) are a growing family of small non-protein-coding regulatory genes that regulate the expression of homologous target-gene transcripts. They have been implicated in the control of cell death and proliferation in flies, haematopoietic lineage differentiation in mammals, neuronal patterning in nematodes and leaf and flower development in plants. miRNAs are processed by the RNA-mediated interference machinery. Drosha is an RNase III enzyme that was recently implicated in miRNA processing. Here we show that human Drosha is a component of two multi-protein complexes. The larger complex contains multiple classes of RNA-associated proteins including RNA helicases, proteins that bind double-stranded RNA, novel heterogeneous nuclear ribonucleoproteins and the Ewing's sarcoma family of proteins. The smaller complex is composed of Drosha and the double-stranded-RNA-binding protein, DGCR8, the product of a gene deleted in DiGeorge syndrome. In vivo knock-down and in vitro reconstitution studies revealed that both components of this smaller complex, termed Microprocessor, are necessary and sufficient in mediating the genesis of miRNAs from the primary miRNA transcript.

MicroRNAs (miRNAs) are generated by a two-step processing pathway to yield RNA molecules of approximately 22 nucleotides that negatively regulate target gene expression at the posttranscriptional level 1 . Primary miRNAs are processed to precursor miRNAs (pre-miRNAs) by the Microprocessor complex2 -4. These pre-miRNAs are cleaved by the RNase III Dicer5 -8 to generate mature miRNAs that direct the RNA-induced silencing complex (RISC) to messenger RNAs with complementary sequence9. Here we show that TRBP (the human immunodeficiency virus transactivating response RNA-binding protein10), which contains three double-stranded, RNAbinding domains, is an integral component of a Dicer-containing complex. Biochemical analysis of TRBP-containing complexes revealed the association of Dicer-TRBP with Argonaute 2 (Ago2)11 , 12, the catalytic engine of RISC. The physical association of Dicer-TRBP and Ago2 was confirmed after the isolation of the ternary complex using Flag-tagged Ago2 cell lines. In vitro reconstitution assays demonstrated that TRBP is required for the recruitment of Ago2 to the small interfering RNA (siRNA) bound by Dicer. Knockdown of TRBP results in destabilization of Dicer and a consequent loss of miRNA biogenesis. Finally, depletion of the Dicer-TRBP complex via exogenously introduced siRNAs diminished RISC-mediated reporter gene silencing. These results support a role of the Dicer-TRBP complex not only in miRNA processing but also as a platform for RISC assembly.To gain an insight into the components of the miRNA/siRNA processing machinery, we isolated a Dicer-containing complex from human cells. This was accomplished by developing HEK293-derived stable cell lines expressing Dicer tagged with Flag (Flag-Dicer). Flag-Dicer was isolated using affinity chromatography, and the affinity eluate was subjected to SDSpolyacrylamide gel electrophoresis (PAGE) followed by silver staining and western blot analysis.Western blot and mass spectrometric analyses indicated that most polypeptides in the Dicer affinity eluate were products of the proteolytic break down of Dicer (Fig. 1a). However, mass spectroscopy identified a 50-kDa band (six peptide sequences that migrated slightly above the contaminating MEP50 band) corresponding to the human immunodeficiency virus (HIV)-1 transactivating response (TAR) RNA-binding protein (TRBP) 10 . The TRBP gene encodes a protein with three double-stranded RNA-binding domains (dsRBDs). Analysis of the nonredundant protein database by Blast identified proteins with close homology to TRBP in both vertebrates and Drosophila (CG6866) (Fig. 1b) (Fig. 1c). The presence of Dicer was also confirmed by mass spectrometric sequencing. Moreover, additional bands (indicated with an asterisk in Fig. 1c) correspond to SKB1 and MEP50, common contaminants of Flag purification. Although most of TRBP eluted in smaller fractions (32 and beyond; perhaps as a consequence of overexpression), a minor portion of TRBP eluted as a large complex (fractions 16 and 18) not easily visualized by silver sta...

RNA interference is implemented through the action of the RNA-induced silencing complex (RISC). Although Argonaute2 has been identified as the catalytic center of RISC, the RISC polypeptide composition and assembly using short interfering RNA (siRNA) duplexes has remained elusive. Here we show that RISC is composed of Dicer, the double-stranded RNA binding protein TRBP, and Argonaute2. We demonstrate that this complex can cleave target RNA using precursor microRNA (pre-miRNA) hairpin as the source of siRNA. Although RISC can also utilize duplex siRNA, it displays a nearly 10-fold greater activity using the pre-miRNA Dicer substrate. RISC distinguishes the guide strand of the siRNA from the passenger strand and specifically incorporates the guide strand. Importantly, ATP is not required for miRNA processing, RISC assembly, or multiple rounds of target-RNA cleavage. These results define the composition of RISC and demonstrate that miRNA processing and target-RNA cleavage are coupled.

We have previously described a multiprotein complex termed the BHC or BRAF-HDAC complex, which is required for the repression of neuronal-specific genes. We have shown that the BHC complex is recruited by a neuronal silencer, REST (RE1-silencing transcription factor), and mediates the repression of REST-responsive genes. BHC is a multiprotein complex consisting of two enzymatic activities: a histone deacetylase (HDAC1 or 2) and a recently described histone demethylase (BHC110, also known as LSD1 or AOF2). Here we show that BHC110-containing complexes show a nearly fivefold increase in demethylation of histone H3 lysine 4 (H3K4) compared to recombinant BHC110. Furthermore, recombinant BHC110 is unable to demethylate H3K4 on nucleosomes, but BHC110-containing complexes readily demethylate nucleosomes. In vitro reconstitution of the BHC complex using recombinant subunits reveals an essential role for the REST corepressor CoREST, not only in stimulating demethylation on core histones but also promoting demethylation of nucleosomal substrates. We find that nucleosomal demethylation is the result of CoREST enhancing the association between BHC110 and nucleosomes. Depletion of CoREST in in vivo cell culture results in de-repression of REST-responsive gene expression and increased methylation of H3K4. Together, these results highlight an essential role for CoREST in demethylation of H3K4 both in vitro and in vivo.

The C-terminal domain (CTD) of RNA polymerase II (RNAPII) is an essential component of transcriptional regulation and RNA processing of protein-coding genes. A large body of data also implicates the CTD in the transcription and processing of RNAPII-mediated small nuclear RNAs (snRNAs). However, the identity of the complex (or complexes) that associates with the CTD and mediates the processing of snRNAs has remained elusive. Here, we describe an RNA polymerase II complex that contains at least 12 novel subunits, termed the Integrator, in addition to core RNAPII subunits. Two of the Integrator subunits display similarities to the subunits of the cleavage and polyadenylation specificity factor (CPSF) complex. We show that Integrator is recruited to the U1 and U2 snRNA genes and mediates the snRNAs' 3' end processing. The Integrator complex is evolutionarily conserved in metazoans and directly interacts with the C-terminal domain of the RNA polymerase II largest subunit.