The Spliceosome: Design Principles of a Dynamic RNP Machine

RNA metabolism, broadly defined as the compendium of all processes that involve RNA, including transcription, processing and modification of transcripts, translation, RNA degradation and its regulation, is the central and most evolutionarily conserved part of cell physiology. A comprehensive, genome-wide census of all enzymatic and non-enzymatic protein domains involved in RNA metabolism was conducted by using sequence profile analysis and structural comparisons. Proteins related to RNA metabolism comprise from 3 to 11% of the complete protein repertoire in bacteria, archaea and eukaryotes, with the greatest fraction seen in parasitic bacteria with small genomes. Approximately one-half of protein domains involved in RNA metabolism are present in most, if not all, species from all three primary kingdoms and are traceable to the last universal common ancestor (LUCA). The principal features of LUCA’s RNA metabolism system were reconstructed by parsimony-based evolutionary analysis of all relevant groups of orthologous proteins. This reconstruction shows that LUCA possessed not only the basal translation system, but also the principal forms of RNA modification, such as methylation, pseudouridylation and thiouridylation, as well as simple mechanisms for polyadenylation and RNA degradation. Some of these ancient domains form paralogous groups whose evolution can be traced back in time beyond LUCA, towards low-specificity proteins, which probably functioned as cofactors for ribozymes within the RNA world framework. The main lineage-specific innovations of RNA metabolism systems were identified. The most notable phase of innovation in RNA metabolism coincides with the advent of eukaryotes and was brought about by the merge of the archaeal and bacterial systems via mitochondrial endosymbiosis, but also involved emergence of several new, eukaryote-specific RNA-binding domains. Subsequent, vast expansions of these domains mark the origin of alternative splicing in animals and probably in plants. In addition to the reconstruction of the evolutionary history of RNA metabolism, this analysis produced numerous functional predictions, e.g. of previously undetected enzymes of RNA modification.

Comparative genomics and evolution of proteins involved in RNA metabolism

The spliceosome removes introns from messenger RNA precursors (pre-mRNA). Decades of biochemistry and genetics combined with recent structural studies of the spliceosome have produced a detailed view of the mechanism of splicing. In this review, we aim to make this mechanism understandable and provide several videos of the spliceosome in action to illustrate the intricate choreography of splicing. The U1 and U2 small nuclear ribonucleoproteins (snRNPs) mark an intron and recruit the U4/U6.U5 tri-snRNP. Transfer of the 5' splice site (5'SS) from U1 to U6 snRNA triggers unwinding of U6 snRNA from U4 snRNA. U6 folds with U2 snRNA into an RNA-based active site that positions the 5'SS at two catalytic metal ions. The branch point (BP) adenosine attacks the 5'SS, producing a free 5' exon. Removal of the BP adenosine from the active site allows the 3'SS to bind, so that the 5' exon attacks the 3'SS to produce mature mRNA and an excised lariat intron.

RNA Splicing by the Spliceosome

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.

Pre-messenger RNA (pre-mRNA) splicing is a central step in gene expression. Lying between transcription and protein synthesis, pre-mRNA splicing removes sequences (introns) that would otherwise disrupt the coding potential of intron-containing transcripts. This process takes place in the nucleus, catalyzed by a large RNA–protein complex called the spliceosome. Prp8p, one of the largest and most highly conserved of nuclear proteins, occupies a central position in the catalytic core of the spliceosome, and has been implicated in several crucial molecular rearrangements that occur there. Recently, Prp8p has also come under the spotlight for its role in the inherited human disease, Retinitis Pigmentosa. Prp8 is unique, having no obvious homology to other proteins; however, using bioinformatical analysis we reveal the presence of a conserved RNA recognition motif (RRM), an MPN/JAB domain and a putative nuclear localization signal (NLS). Here, we review biochemical and genetical data, mostly related to the human and yeast proteins, that describe Prp8’s central role within the spliceosome and its molecular interactions during spliceosome formation, as splicing proceeds, and in post-splicing complexes.

/pdf/prp8-protein-at-the-heart-of-the-spliceosome-4a61tzvfi6.pdf

Prp8 protein: At the heart of the spliceosome

Prp2p, Prp16p, Prp22p, and Prp43p are members of the DEAH-box family of ATP-dependent putative RNA helicases required for pre-mRNA splicing in Saccharomyces cerevisiae. Recently, mammalian homologues of Prp43p and Prp22p have been described, supporting the idea that splicing in yeast and man is phylogenetically conserved. In this study, we show that a murine cell line resistant to the novel immunoregulatory drug Leflunomide (Arava TM ) overexpresses a 135-kDa protein that is a putative DEAH-box RNA helicase. We have cloned the human counterpart of this protein and show that it shares pronounced sequence homology with Prp16p. Apart from its N-terminal domain, which is rich in RS, RD, and RE dipeptides, this human homologue of Prp16p (designated hPrp16p) is 41% identical to Prp16p. Significantly, homology is not only observed within the phylogenetically conserved helicase domain, but also in Prp16p-specific sequences. Immunofluorescence microscopy studies demonstrated that hPrp16p co-localizes with snRNPs in subnuclear structures referred to as speckles. Antibodies specific for hPrp16p inhibited pre-mRNA splicing in vitro prior to the second step. Thus, like its yeast counterpart, hPrp16p also appears to be required for the second catalytic step of splicing. Taken together, our data indicate that the human 135-kDa protein identified here is the structural and functional homologue of the yeast putative RNA helicase, Prp16p.

/pdf/the-mammalian-homologue-of-prp16p-is-overexpressed-in-a-cell-1iltv8zedn.pdf

The mammalian homologue of Prp16p is overexpressed in a cell line tolerant to Leflunomide, a new immunoregulatory drug effective against rheumatoid arthritis.

The driving forces behind the many RNA conformational changes occurring in the spliceosome are not well understood. Here we characterize an evolutionarily conserved human U5 small nuclear ribonucleoprotein (snRNP) protein (U5‐116kD) that is strikingly homologous to the ribosomal elongation factor EF‐2 (ribosomal translocase). A 114 kDa protein (Snu114p) homologous to U5‐116kD was identified in Saccharomyces cerevisiae and was shown to be essential for yeast cell viability. Genetic depletion of Snu114p results in accumulation of unspliced pre‐mRNA, indicating that Snu114p is essential for splicing in vivo. Antibodies specific for U5‐116kD inhibit pre‐mRNA splicing in a HeLa nuclear extract in vitro . In HeLa cells, U5‐116kD is located in the nucleus and co‐localizes with snRNP‐containing subnuclear structures referred to as speckles. The G domain of U5‐116kD/Snu114p contains the consensus sequence elements G1–G5 important for binding and hydrolyzing GTP. Consistent with this, U5‐116kD can be cross‐linked specifically to GTP by UV irradiation of U5 snRNPs. Moreover, a single amino acid substitution in the G1 sequence motif of Snu114p, expected to abolish GTP‐binding activity, is lethal, suggesting that GTP binding and probably GTP hydrolysis is important for the function of U5‐116kD/Snu114p. This is to date the first evidence that a G domain‐containing protein plays an essential role in the pre‐mRNA splicing process.

An evolutionarily conserved U5 snRNP-specific protein is a GTP-binding factor closely related to the ribosomal translocase EF-2.

Splicing of nuclear precursors of mRNA (pre-mRNA) involves dynamic interactions between the RNA constituents of the spliceosome. The rearrangement of RNA–RNA interactions, such as the unwinding of the U4/U6 duplex, is believed to be driven by ATP-dependent RNA helicases. We recently have shown that spliceosomal U5 small nuclear ribonucleoproteins (snRNPs) from HeLa cells contain two proteins, U5–200kD and U5–100kD, which share homology with the DEAD/DEXH-box families of RNA helicases. Here we demonstrate that purified U5 snRNPs exhibit ATP-dependent unwinding of U4/U6 RNA duplices in vitro. To identify the protein responsible for this activity, U5 snRNPs were depleted of a subset of proteins under high salt concentrations and assayed for RNA unwinding. The activity was retained in U5 snRNPs that contain the U5–200kD protein but lack U5–100kD, suggesting that the U5–200kD protein could mediate U4/U6 duplex unwinding. Finally, U5–200kD was purified to homogeneity by glycerol gradient centrifugation of U5 snRNP proteins in the presence of sodium thiocyanate, followed by ion exchange chromatography. The RNA unwinding activity was found to reside exclusively with the U5–200kD DEXH-box protein. Our data raise the interesting possibility that this RNA helicase catalyzes unwinding of the U4/U6 RNA duplex in the spliceosome.

The human U5-200kD DEXH-box protein unwinds U4/U6 RNA duplices in vitro.

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The mammalian homologue of Prp16p is overexpressed in a cell line tolerant to Leflunomide, a new immunoregulatory drug effective against rheumatoid arthritis.

An evolutionarily conserved U5 snRNP-specific protein is a GTP-binding factor closely related to the ribosomal translocase EF-2.

The human U5-200kD DEXH-box protein unwinds U4/U6 RNA duplices in vitro.