The precision and complexity of intron removal during pre-mRNA splicing still amazes even 26 years after the discovery that the coding information of metazoan genes is interrupted by introns (Berget et al. 1977; Chow et al. 1977). Adding to this amazement is the recent realization that most human genes express more than one mRNA by alternative splicing, a process by which functionally diverse protein isoforms can be expressed according to different regulatory programs. Given that the vast majority of human genes contain introns and that most pre-mRNAs undergo alternative splicing, it is not surprising that disruption of normal splicing patterns can cause or modify human disease. The purpose of this review is to highlight the different mechanisms by which disruption of pre-mRNA splicing play a role in human disease. Several excellent reviews provide detailed information on splicing and the regulation of splicing (Burge et al. 1999; Hastings and Krainer 2001; Black 2003). The potential role of splicing as a modifier of human disease has also recently been reviewed (NissimRafinia and Kerem 2002).

http://genesdev.cshlp.org/content/17/4/419.full.pdf

Pre-mRNA splicing and human disease

Spliceosomal UsnRNP biogenesis, structure and function.

f Abstract Ubiquitous in eukaryotic cells, the La protein associates with the 3 termini of many newly synthesized small RNAs. RNAs bound by the La protein include all nascent transcripts made by RNA polymerase III as well as certain small RNAs synthesized by other RNA polymerases. Recent genetic and biochemical analyses have revealed that binding by the La protein protects the 3 ends of these RNAs from exonucleases. This La-mediated stabilization is required for the normal pathway of pre-tRNA maturation, facilitates assembly of small RNAs into functional RNA-protein complexes, and contributes to nuclear retention of certain small RNAs. Studies of mutant La proteins have given some insights into how the La protein specifically recognizes its RNA targets. However, many questions remain regarding the molecular mechanisms by which La protein binding influences multiple steps in small RNA biogenesis. This review focuses on the roles of the La protein in small RNA biogenesis and also discusses data that implicate the La protein in the translation of specific mRNAs.

The la protein

Sm and Sm-like (LSm) proteins form heptameric complexes that are involved in various steps of RNA metabolism. In yeast, the Lsm1-7 complex functions in mRNA degradation and is associated with several enzymes of this pathway, while the complex LSm2-8, the composition of which largely overlaps with that of LSm1-7, has a role in pre-mRNA splicing. A human gene encoding an LSm1 homolog has been identified, but its role in mRNA degradation has yet to be elucidated. We performed subcellular localization studies and found hLSm1 predominantly in the cytoplasm. However, it is not distributed evenly; rather, it is highly enriched in small, discrete foci. The endogenous hLSm4 is similarly localized, as are the overexpressed proteins hLSm1-7, but not hLSm8. The foci also contain two key factors in mRNA degradation, namely the decapping enzyme hDcp1/2 and the exonuclease hXrn1. Moreover, coexpression of wild-type and mutant LSm proteins, as well as fluorescence resonance energy transfer (FRET) studies, indicate that the mammalian proteins hLSm1-7 form a complex similar to the one found in yeast, and that complex formation is required for enrichment of the proteins in the cytoplasmic foci. Therefore, the foci contain a partially or fully assembled machinery for the degradation of mRNA.

The human LSm1-7 proteins colocalize with the mRNA-degrading enzymes Dcp1/2 and Xrn1 in distinct cytoplasmic foci

In eukaryotes, seven Sm proteins bind to the U1, U2, U4 and U5 spliceosomal snRNAs while seven Smlike proteins (Lsm2p–Lsm8p) are associated with U6 snRNA Another yeast Sm-like protein, Lsm1p, does not interact with U6 snRNA Surprisingly, using the tandem affinity purification (TAP) method, we identified Lsm1p among the subunits associated with Lsm3p Coprecipitation experiments demonstrated that Lsm1p, together with Lsm2p–Lsm7p, forms a new sevensubunit complex We purified the two related Sm-like protein complexes and identified the proteins recovered in the purified preparations by mass spectrometry This confirmed the association of the Lsm2p–Lsm8p complex with U6 snRNA In contrast, the Lsm1p–Lsm7p complex is associated with Pat1p and Xrn1p exoribonuclease, suggesting a role in mRNA degradation Deletions of LSM1, 6, 7 and PAT1 genes increased the half-life of reporter mRNAs Interestingly, accumulating mRNAs were capped, suggesting a block in mRNA decay at the decapping step These results indicate the involvement of a new conserved Sm-like protein complex and a new factor, Pat1p, in mRNA degradation and suggest a physical connection between decapping and exonuclease trimming

A Sm-like protein complex that participates in mRNA degradation.

Through a combination of in vitro snRNP reconstitution, photocross-linking and immunoprecipitation techniques, we have investigated the interaction of proteins with the spliceosomal U6 snRNA in U6 snRNPs, U4/U6 di-snRNPs and U4/U6.U5 tri-snRNPs. Of the seven Lsm (Sm-like) proteins that associate specifically with this spliceosomal snRNA, three were shown to contact the RNA directly, and to maintain contact as the U6 RNA is incorporated into tri-snRNPs. In tri-snRNPs, the U5 snRNP protein Prp8 contacts position 54 of U6, which is in the conserved region that contributes to the formation of the catalytic core of the spliceosome. Other tri-snRNP-specific contacts were also detected, indicating the dynamic nature of protein interactions with this important snRNA. The uridine-rich extreme 39 end of U6 RNA was shown to be essential but not sufficient for the association of the Lsm proteins. Interestingly, the Lsm proteins associate efficiently with the 39 half of U6, which contains the 39 stem-loop and uridine-rich 39 end, suggesting that the Lsm and Sm proteins may recognize similar features in RNAs.

Valerie P.I. Vidal

Papers

Characterization of U6 snRNA-protein interactions.