One of the most amazing findings in molecular biology was the discovery that eukaryotic genes are discontinuous, interrupted by stretches of non-coding sequence. The subsequent realization that the intervening regions are removed from pre-mRNA transcripts via the activity of a common set of small nuclear RNAs (snRNAs), which assemble together with associated proteins into a spliceosome, was equally surprising. How do cells orchestrate the assembly of this molecular machine? And how does the spliceosome accurately recognize exons and introns to carry out the splicing reaction? Insights into these questions have been gained by studying the life cycle of spliceosomal snRNAs from their transcription, nuclear export and reimport, all the way through to their dynamic assembly into the spliceosome. This assembly process can also affect the regulation of alternative splicing and has implications for human disease.

Recent advances have fuelled rapid growth in our appreciation of the tremendous number, diversity and biological importance of non-coding (nc)RNAs. Because ncRNAs typically function as ribonucleoprotein (RNP) complexes and not as naked RNAs, understanding their biogenesis is crucial to comprehending their regulation and function. The small nuclear and small nucleolar RNPs are two well studied classes of ncRNPs with elaborate assembly and trafficking pathways that provide paradigms for understanding the biogenesis of other ncRNPs.

The D-type cyclin-dependent kinases CDK4 and CDK6 are complexed with many small cellular proteins (p14, plS, p16, plS, and p20). We have isolated cDNA sequences corresponding to the MTS2 genomic fragment that encodes the CDK4-and CDK6-associated p14 protein. By use of a yeast interaction screen to search for CDK6-interacting proteins, we have also identified an 18-kD human protein, p18, that is a homolog of the cyclin D-CDK4 inhibitors p16 {INK4A/MTS1) and p14 (MTS2/INK4B). Both in vivo and in vitro, p18 interacts strongly with CDK6, weakly with CDK4, and exhibits no detectable interaction with the other known CDKs. Recombinant p18 inhibits the kinase activity of cyclin D-CDK6. Distinct from the p21/p27 family of CDK inhibitors that form ternary complexes with cyclin-CDKs, only binary complexes of p14, p16, and p18 were found in association with CDK4 and/or CDK6. Ectopic expression of p18 or p16 suppresses cell growth with a correlated dependence on endogenous wild-type pRb.[Key Words: Cyclin-dependent kinase inhibitors; cell cycle; CDK4 and CDK6 interacting proteins]

In eukaryotic cells, histone gene expression is one of the major events that mark entry into S phase. While this process is tightly linked to cell cycle position, how it is regulated by the cell cycle machinery is not known. Here we show that NPAT, a substrate of the cyclin E-Cdk2 complex, is associated with human replication-dependent histone gene clusters on both chromosomes 1 and 6 in S phase. We demonstrate that NPAT activates histone gene transcription and that this activation is dependent on the promoter elements (SSCSs) previously proposed to mediate cell cycle-dependent transcription. Cyclin E is also associated with the histone gene loci, and cyclin E-Cdk2 stimulates the NPAT-mediated activation of histone gene transcription. Thus, our results both show that NPAT is involved in a key S phase event and provide a link between the cell cycle machinery and activation of histone gene transcription.

Cajal bodies (CBs) are nuclear suborganelles involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs). In addition to snRNPs, they are highly enriched in basal transcription and cell cycle factors, the nucleolar proteins fibrillarin (Fb) and Nopp140 (Nopp), the survival motor neuron (SMN) protein complex, and the CB marker protein, p80 coilin. We report the generation of knockout mice lacking the COOH-terminal 487 amino acids of coilin. Northern and Western blot analyses demonstrate that we have successfully removed the full-length coilin protein from the knockout animals. Some homozygous mutant animals are viable, but their numbers are reduced significantly when crossed to inbred backgrounds. Analysis of tissues and cell lines from mutant animals reveals the presence of extranucleolar foci that contain Fb and Nopp but not other typical nucleolar markers. These so-called “residual” CBs neither condense Sm proteins nor recruit members of the SMN protein complex. Transient expression of wild-type mouse coilin in knockout cells results in formation of CBs and restores these missing epitopes. Our data demonstrate that full-length coilin is essential for proper formation and/or maintenance of CBs and that recruitment of snRNP and SMN complex proteins to these nuclear subdomains requires sequences within the coilin COOH terminus.

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Coiled bodies (CBs) are nuclear organelles whose structures appear to be highly conserved in evolution.In rapidly cycling cells, they are typically located in the nucleoplasm but are often found in contact with the nucleolus. The CBs in human cells contain a unique protein, called p80-coilin. Studies on amphibian oocyte nuclei have revealed a protein within the "sphere" organelle that shares significant structural similarity to p80-coilin. Eukaryotic cells can be viewed as if they were a mixture of compartments, some of which can be defined physically, some functionally. In the cytoplasmic compartment, the roles of the various organelles and the interplay between them have been extensively studied. In contrast, the domains and their interconnections within the nucleus are poorly understood. However, it is now clear that the nucleus is a highly organized structure, permeated by a proteinaceous "matrix" and composed of many different subdomains. The so-called interchromatin space of somatic cells can be characterized at the ultrastructural level by several types of structures. These are perichromatin fibrils, interchromatin granule clusters, and nuclear bodies. Small nuclear ribonucleoproteins (snRNPs) are major components of each of these three subcompartments (see refs. 1 and 2 for reviews). One type of nuclear body, the coiled body (CB), contains relatively high concentrations of small RNPs as well as other proteins (see Table 1 for a list of components reported to be either enriched or not enriched in CBs). Despite (or perhaps because of) this rather lengthy roster, the list of putative CB functions is nearly as long (see refs. 3 and 4 for reviews). Early studies on amphibian oocyte nuclei also revealed morphologically distinct structures, including: nucleoli, lampbrush chromosomes, and "spheres." The subsequent finding that spheres (which are composed of B-and C-type "snurposomes") contain numerous snRNPs and snRNP proteins caused speculation that CBs and spheres were related organelles (reviewed in ref. 5). The final pieces to the puzzle linking CBs and spheres were revealed when SPH-1, an integral protein component ofThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.C snurposomes, was shown to have significant structural homology to a CB marker protein, p80-coilin (6, 7), and when Wu et al (8) showed that p80-coilin protein is targeted to spheres when the human coilin mRNA was injected into amphibian oocytes. Thus spheres and CBs are homologous organelles that are likely to have similar functions, but as yet no functions have been demonstrated (5).Of particular interest is the fact that some of the spheres within the amphibian oocyte are attached to the so-called "sphere-organizer" regions of the lampbrush chromosomes. These sphere-organizers are located at the histone gene clusters in two classes of amphibia, Anura and Urodela (9, 10). The fa...

Spinal muscular atrophy (SMA) is a genetic disorder caused by mutations in the human survival of motor neuron 1 gene, SMN1. SMN protein is part of a large complex that is required for biogenesis of various small nuclear ribonucleoproteins (snRNPs). Here, we report that SMN interacts directly with the Cajal body signature protein, coilin, and that this interaction mediates recruitment of the SMN complex to Cajal bodies. Mutation or deletion of specific RG dipeptide residues within coilin inhibits the interaction both in vivo and in vitro. Interestingly, GST-pulldown experiments show that coilin also binds directly to SmB. Competition studies show that coilin competes with SmB for binding sites on SMN. Ectopic expression of SMN and coilin constructs in mouse embryonic fibroblasts lacking endogenous coilin confirms that recruitment of SMN and splicing snRNPs to Cajal bodies depends on the coilin C-terminal RG motif. A cardinal feature of SMA patient cells is a defect in the targeting of SMN to nuclear foci; our results uncover a role for coilin in this process.