Characterization of DGCR8/Pasha, the essential cofactor for Drosha in primary miRNA processing

DGCR8/Pasha is an essential cofactor for Drosha, a nuclear RNase III that cleaves the local hairpin structures embedded in long primary microRNA transcripts (pri-miRNAs) in eukaryotes. Although our knowledge of pri-miRNA processing has significantly advanced in recent years, the precise role of DGCR8 in this pathway remains unclear. In our present study, we dissect the domains in DGCR8 that contribute to the processing of pri-miRNAs and the subcellular localization of DGCR8. Drosha is stabilized through an interaction between its middle domain and the conserved C-terminal domain of DGCR8. Furthermore, DGCR8, but not Drosha, can directly and stably interact with pri-miRNAs, and the tandem dsRNA-binding domains (dsRBDs) in DGCR8 are responsible for this recognition. Moreover, the DGCR8 N-terminal region upstream of its dsRBDs is unnecessary for pri-miRNA processing but is critical for nuclear localization. Our study thus provides further insights into the mechanism of action of the Drosha-DGCR8 complex in pri-miRNA processing

Single-molecule approach to immunoprecipitated protein complexes: Insights into miRNA uridylation

Single-molecule techniques have been used for only a subset of biological problems because of difficulties in studying proteins that require cofactors or post-translational modifications. Here, we present a new method integrating single-molecule fluorescence microscopy and immunopurification to study protein complexes. We used this method to investigate Lin28-mediated microRNA uridylation by TUT4 (terminal uridylyl transferase 4, polyU polymerase), which regulates let-7 microRNA biogenesis. Our real-time analysis of the uridylation by the TUT4 immunoprecipitates suggests that Lin28 functions as a processivity factor of TUT4. Our new technique, SIMPlex (single-molecule approach to immunoprecipitated protein complexes), provides a universal tool to analyse complex proteins at the single-molecule level

Multiple determinants of splicing repression activity in the polypyrimidine tract binding proteins, PTBP1 and PTBP2

Most human genes generate multiple protein isoforms through alternative pre-mRNA splicing, but the mechanisms controlling alternative splicing choices by RNA binding proteins are not well understood. These proteins can have multiple paralogs expressed in different cell types and exhibiting different splicing activities on target exons. We examined the paralogous polypyrimidine tract binding proteins PTBP1 and PTBP2 to understand how PTBP1 can exhibit greater splicing repression activity on certain exons. Using both an in vivo coexpression assay and an in vitro splicing assay, we show that PTBP1 is more repressive than PTBP2 per unit protein on a target exon. Constructing chimeras of PTBP1 and 2 to determine amino acid features that contribute to their differential activity, we find that multiple segments of PTBP1 increase the repressive activity of PTBP2. Notably, when either RRM1 of PTBP2 or the linker peptide separating RRM2 and RRM3 are replaced with the equivalent PTBP1 sequences, the resulting chimeras are highly active for splicing repression. These segments are distinct from the known region of interaction for the PTBP1 cofactors Raver1 and Matrin3 in RRM2. We find that RRM2 of PTBP1 also increases the repression activity of an otherwise PTBP2 sequence, and that this is potentially explained by stronger binding by Raver1. These results indicate that multiple features over the length of the two proteins affect their ability to repress an exon

Multiple determinants of splicing repression activity in the polypyrimidine tract binding proteins, PTBP1 and PTBP2

Most human genes generate multiple protein isoforms through alternative pre-mRNA splicing, but the mechanisms controlling alternative splicing choices by RNA binding proteins are not well understood. These proteins can have multiple paralogs expressed in different cell types and exhibiting different splicing activities on target exons. We examined the paralogous polypyrimidine tract binding proteins PTBP1 and PTBP2 to understand how PTBP1 can exhibit greater splicing repression activity on certain exons. Using both an in vivo coexpression assay and an in vitro splicing assay, we show that PTBP1 is more repressive than PTBP2 per unit protein on a target exon. Constructing chimeras of PTBP1 and 2 to determine amino acid features that contribute to their differential activity, we find that multiple segments of PTBP1 increase the repressive activity of PTBP2. Notably, when either RRM1 of PTBP2 or the linker peptide separating RRM2 and RRM3 are replaced with the equivalent PTBP1 sequences, the resulting chimeras are highly active for splicing repression. These segments are distinct from the known region of interaction for the PTBP1 cofactors Raver1 and Matrin3 in RRM2. We find that RRM2 of PTBP1 also increases the repression activity of an otherwise PTBP2 sequence, and that this is potentially explained by stronger binding by Raver1. These results indicate that multiple features over the length of the two proteins affect their ability to repress an exon.National Institutes of Health Ruth L. Kirschstein National Research Service Award (NIH-NRSA) [1F32GM093533]; CSUPERB New Investigator Award; NIH [R01GM049662, R21CA170786]; UCLA Broad Stem Cell Research Center's Training Program12 month embargo; published online: 10 June 2016This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Crystal structure of human DGCR8 core

A complex of Drosha with DGCR8 ( or its homolog Pasha) cleaves primary microRNA ( pri- miRNA) substrates into precursor miRNA and initiates the microRNA maturation process. Drosha provides the catalytic site for this cleavage, whereas DGCR8 or Pasha provides a frame for anchoring substrate pri- miRNAs. To clarify the molecular basis underlying recognition of pri- miRNA by DGCR8 and Pasha, we determined the crystal structure of the human DGCR8 core ( DGCR8S, residues 493 - 720). In the structure, the two double- stranded RNA - binding domains ( dsRBDs) are arranged with pseudo two- fold symmetry and are tightly packed against the C- terminal helix. The H2 helix in each dsRBD is important for recognition of pri- miRNA substrates. This structure, together with fluorescent resonance energy transfer and mutational analyses, suggests that the DGCR8 core recognizes pri- miRNA in two possible orientations. We propose a model for DGCR8' s recognition of pri- miRNA

Tracking pre-mRNA maturation across subcellular compartments identifies developmental gene regulation through intron retention and nuclear anchoring

Steps of mRNA maturation are important gene regulatory events that occur in distinct cellular locations. However, transcriptomic analyses often lose information on the subcellular distribution of processed and unprocessed transcripts. We generated extensive RNA-seq data sets to track mRNA maturation across subcellular locations in mouse embryonic stem cells, neuronal progenitor cells, and postmitotic neurons. We find disparate patterns of RNA enrichment between the cytoplasmic, nucleoplasmic, and chromatin fractions, with some genes maintaining more polyadenylated RNA in chromatin than in the cytoplasm. We bioinformatically defined four regulatory groups for intron retention, including complete cotranscriptional splicing, complete intron retention in the cytoplasmic RNA, and two intron groups present in nuclear and chromatin transcripts but fully excised in cytoplasm. We found that introns switch their regulatory group between cell types, including neuronally excised introns repressed by polypyrimidine track binding protein 1 (PTBP1). Transcripts for the neuronal gamma-aminobutyric acid (GABA) B receptor, 1 (Gabbr1) are highly expressed in mESCs but are absent from the cytoplasm. Instead, incompletely spliced Gabbr1 RNA remains sequestered on chromatin, where it is bound by PTBP1, similar to certain long noncoding RNAs. Upon neuronal differentiation, Gabbr1 RNA becomes fully processed and exported for translation. Thus, splicing repression and chromatin anchoring of RNA combine to allow posttranscriptional regulation of Gabbr1 over development. For this and other genes, polyadenylated RNA abundance does not indicate functional gene expression. Our data sets provide a rich resource for analyzing many other aspects of mRNA maturation in subcellular locations and across development