Molecular architecture of the Saccharomyces cerevisiae activated spliceosome.

The activated spliceosome (Bact) is in a catalytically inactive state and is remodeled into a catalytically active machine by the RNA helicase Prp2, but the mechanism is unclear. Here, we describe a 3D electron cryomicroscopy structure of the Saccharomyces cerevisiae Bact complex at 5.8-angstrom resolution. Our model reveals that in Bact, the catalytic U2/U6 RNA-Prp8 ribonucleoprotein core is already established, and the 5' splice site (ss) is oriented for step 1 catalysis but occluded by protein. The first-step nucleophile-the branchsite adenosine-is sequestered within the Hsh155 HEAT domain and is held 50 angstroms away from the 5'ss. Our structure suggests that Prp2 adenosine triphosphatase-mediated remodeling leads to conformational changes in Hsh155's HEAT domain that liberate the first-step reactants for catalysis.

The network of protein-protein interactions within the human U4/U6.U5 tri-snRNP.

The human 25S U4/U6.U5 tri-snRNP is a major building block of the U2-type spliceosome and contains, in addition to the U4, U6, and U5 snRNAs, at least 30 distinct proteins. To learn more about the molecular architecture of the tri-snRNP, we have investigated interactions between tri-snRNP proteins using the yeast two-hybrid assay and in vitro binding assays, and, in addition, have identified distinct protein domains that are critical for the connectivity of this protein network in the human tri-snRNP. These studies revealed multiple interactions between distinct domains of the U5 proteins hPrp8, hBrr2 (a DExH/D-box helicase), and hSnu114 (a putative GTPase), which are key players in the catalytic activation of the spliceosome, during which the U4/U6 base-pairing interaction is disrupted and U4 is released from the spliceosome. Both the U5-specific, TPR/HAT-repeat-containing hPrp6 protein and the tri-snRNP-specific hSnu66 protein interact with several U5- and U4/U6-associated proteins, including hBrr2 and hPrp3, which contacts the U6 snRNA. Thus, both proteins are located at the interface between U5 and U4/U6 in the tri-snRNP complex, and likely play an important role in transmitting the activity of hBrr2 and hSnu114 in the U5 snRNP to the U4/U6 duplex during spliceosome activation. A more detailed analysis of these protein interactions revealed that different HAT repeats mediate interactions with specific hPrp6 partners. Taken together, data presented here provide a detailed picture of the network of protein interactions within the human tri-snRNP.

The human 18S U11/U12 snRNP contains a set of novel proteins not found in the U2-dependent spliceosome.

U11 and U12 snRNPs bind U12-type pre-mRNAs as a preformed di-snRNP complex, simultaneously recognizing the 5' splice site and branchpoint sequence. Thus, within the U12-type prespliceosome, U11/U12 components form a molecular bridge connecting both ends of the intron. We have affinity purified human 18S U11/U12 and 12S U11 snRNPs, and identified their protein components by using mass spectrometry. U11/U12 snRNPs lack all known U1 snRNP proteins but contain seven novel proteins (i.e., 65K, 59K, 48K, 35K, 31K, 25K, 20K) not found in the major spliceosome, four of which (59K, 48K, 35K, and 25K) are U11-associated. Thus, protein-protein and protein-RNA interactions contributing to 5' splice site recognition and/or intron bridging appear to differ significantly in the minor versus major prespliceosome. The majority of U11/U12 proteins are highly conserved in organisms known to contain U12-type introns. However, homologs of those associated with U11 were not detected in Drosophila melanogaster, consistent with the presence of a divergent U11 snRNP in flies. RNAi experiments revealed that several U11/U12 proteins are essential for cell viability, suggesting they play key roles in U12-type splicing. The presence of unique U11/U12 snRNP proteins in the U12-type spliceosome provides insight into potential evolutionary relationships between the major and minor spliceosome.

New microRNAs from mouse and human.

MicroRNAs (miRNAs) represent a new class of noncoding RNAs encoded in the genomes of plants, invertebrates, and vertebrates. MicroRNAs regulate translation and stability of target mRNAs based on (partial) sequence complementarity. Although the number of newly identified miRNAs is still increasing, target mRNAs of animal miRNAs remain to be identified. Here we describe 31 novel miRNAs that were identified by cloning from mouse tissues and the human Saos-2 cell line. Fifty-three percent of all known mouse and human miRNAs have homologs in Fugu rubripes (pufferfish) or Danio rerio (zebrafish), of which almost half also have a homolog in Caenorhabditis elegans or Drosophila melanogaster. Because of the recurring identification of already known miRNAs and the unavoidable background of ribosomal RNA breakdown products, it is believed that not many more miRNAs may be identified by cloning. A comprehensive collection of miRNAs is important for assisting bioinformatics target mRNA identification and comprehensive genome annotation.

Identification of tissue-specific microRNAs from mouse.

MicroRNAs (miRNAs) are a new class of noncoding RNAs, which are encoded as short inverted repeats in the genomes of invertebrates and vertebrates. It is believed that miRNAs are modulators of target mRNA translation and stability, although most target mRNAs remain to be identified. Here we describe the identification of 34 novel miRNAs by tissue-specific cloning of approximately 21-nucleotide RNAs from mouse. Almost all identified miRNAs are conserved in the human genome and are also frequently found in nonmammalian vertebrate genomes, such as pufferfish. In heart, liver, or brain, it is found that a single, tissue-specifically expressed miRNA dominates the population of expressed miRNAs and suggests a role for these miRNAs in tissue specification or cell lineage decisions. Finally, a miRNA was identified that appears to be the fruitfly and mammalian ortholog of C. elegans lin-4 stRNA.

Ribonuclease III: new sense from nuisance.

RNases play an important role in the processing of precursor RNAs, creating the mature, functional RNAs. The ribonuclease III family currently is one of the most interesting families of endoribonucleases. Surprisingly, RNase III is involved in the maturation of almost every class of prokaryotic and eukaryotic RNA. We present an overview of the various substrates and their processing. RNase III contains one of the most prominent protein domains used in RNA-protein recognition, the double-stranded RNA binding domain (dsRBD). Progress in the understanding of this domain is summarized. Furthermore, RNase III only recently emerged as a key player in the new exciting biological field of RNA silencing, or RNA interference. The eukaryotic RNase III homologues which are likely involved in this process are compared with the other members of the RNase III family.