Cus2 enforces the first ATP-dependent step of splicing by binding to yeast SF3b1 through a UHM-ULM interaction.

Stable recognition of the intron branchpoint (BP) by the U2 snRNP to form the pre-spliceosome is the first ATP-dependent step of splicing. Genetic and biochemical data from yeast indicate that Cus2 aids U2 snRNA folding into the stem IIa conformation prior to pre-spliceosome formation. Cus2 must then be removed by an ATP-dependent function of Prp5 before assembly can progress. However, the location from which Cus2 is displaced and the nature of its binding to the U2 snRNP are unknown. Here, we show that Cus2 contains a conserved UHM (U2AF homology motif) that binds Hsh155, the yeast homolog of human SF3b1, through a conserved ULM (U2AF ligand motif). Mutations in either motif block binding and allow pre-spliceosome formation without ATP. A 2.0 Å resolution structure of the Hsh155 ULM in complex with the UHM of Tat-SF1, the human homolog of Cus2, and complementary binding assays show that the interaction is highly similar between yeast and humans. Furthermore, we show that Tat-SF1 can replace Cus2 function by enforcing ATP dependence of pre-spliceosome formation in yeast extracts. Cus2 is removed before pre-spliceosome formation, and both Cus2 and its Hsh155 ULM binding site are absent from available cryo-EM structure models. However, our data are consistent with the apparent location of the disordered Hsh155 ULM between the U2 stem-loop IIa and the HEAT repeats of Hsh155 that interact with Prp5. We propose a model in which Prp5 uses ATP to remove Cus2 from Hsh155 such that extended base-pairing between U2 snRNA and the intron BP can occur.

Complex assembly mechanism and an RNA-binding mode of the human p14-SF3b155 spliceosomal protein complex identified by NMR solution structure and functional analyses.

The spliceosomal protein p14, a component of the SF3b complex in the U2 small nuclear ribonucleoprotein (snRNP), is essential for the U2 snRNP to recognize the branch site adenosine. The elucidation of the dynamic process of the splicing machinery rearrangement awaited the solution structural information. We identified a suitable complex of human p14 and the SF3b155 fragment for the determination of its solution structure by NMR. In addition to the overall structure of the complex, which was recently reported in a crystallographic study (typical RNA recognition motif fold beta1-alpha1-beta2-beta3-alpha2-beta4 of p14, and alphaA-betaA fold of the SF3b155 fragment), we identified three important features revealed by the NMR solution structure. First, the C-terminal extension and the nuclear localization signal of p14 (alpha3 and alpha4 in the crystal structure, respectively) were dispensable for the complex formation. Second, the proline-rich segment of SF3b155, following betaA, closely approaches p14. Third, interestingly, the beta1-alpha1 loop and the alpha2-beta4 beta-hairpin form a positively charged groove. Extensive mutagenesis analyses revealed the functional relevance of the residues involved in the protein-protein interactions: two aromatic residues of SF3b155 (Phe408 and Tyr412) play crucial roles in the complex formation, and two hydrophobic residues (Val414 and Leu415) in SF3b 155 serve as an anchor for the complex formation, by cooperating with the aromatic residues. These findings clearly led to the conclusion that SFb155 binds to p14 with three contact points, involving Phe408, Tyr412, and Val414/Leu415. Furthermore, to dissect the interactions between p14 and the branch site RNA, we performed chemical-shift-perturbation experiments, not only for the main-chain but also for the side-chain resonances, for several p14-SF3b155 complex constructs upon binding to RNA. These analyses identified a positively charged groove and the C-terminal extension of p14 as RNA-binding sites. Strikingly, an aromatic residue in the beta1-alpha1 loop, Tyr28, and a positively charged residue in the alpha2-beta4 beta-hairpin, Agr85, are critical for the RNA-binding activity of the positively charged groove. The Tyr28Ala and Arg85Ala point mutants and a deletion mutant of the C-terminal extension clearly revealed that their RNA binding activities were independent of each other. Collectively, this study provides details for the protein-recognition mode of p14 and insight into the branch site recognition.

Ddx42p--a human DEAD box protein with RNA chaperone activities.

The human gene ddx42 encodes a human DEAD box protein highly homologous to the p68 subfamily of RNA helicases. In HeLa cells, two ddx42 poly(A)+ RNA species were detected both encoding the nuclear localized 938 amino acid Ddx42p polypeptide. Ddx42p has been heterologously expressed and its biochemical properties characterized. It is an RNA binding protein, and ATP and ADP modulate its RNA binding affinity. Ddx42p is an NTPase with a preference for ATP, the hydrolysis of which is enhanced by various RNA substrates. It acts as a non-processive RNA helicase. Interestingly, RNA unwinding by Ddx42p is promoted in the presence of a single-strand (ss) binding protein (T4gp32). Ddx42p, particularly in the ADP-bound form (the state after ATP hydrolysis), also mediates efficient annealing of complementary RNA strands thereby displacing the ss binding protein. Ddx42p therefore represents the first example of a human DEAD box protein possessing RNA helicase, protein displacement and RNA annealing activities. The adenosine nucleotide cofactor bound to Ddx42p apparently acts as a switch that controls the two opposing activities: ATP triggers RNA strand separation, whereas ADP triggers annealing of complementary RNA strands.

Complementary deletions in expressed potato U2snRNA gene variants support the hypothesis that stem-loop IIb is dispensable for splicing.

A polymerase chain reaction (PCR) strategy designed to amplify DNA sequences between closely linked U2snRNA genes has generated extensive coding and 5' regulatory sequence information on the potato U2snRNA multigene family. Two of the U2snRNA coding sequences isolated differed substantially from normal U2snRNAs by containing both complementary deletions and regions of novel sequence. However, sequences such as Sm-binding sites and loops of stem-loops III and IV, which are some of the most highly conserved regions in U2snRNA, remain highly conserved in these genes. The complementary deletions would effectively remove stem-loop IIb which has been shown in yeast to be unnecessary for pre-mRNA splicing. Transcripts from one of the genes have been detected by reverse transcriptase-PCR (RT-PCR) in total RNA. These novel U2snRNA genes represent the first reported example of naturally occurring structural variants and provide support for the proposed non-essential role of U2snRNA stem-loop IIb.