Saccharomyces cerevisiae Ecm2 Modulates the Catalytic Steps of pre-mRNA Splicing.

Genetic, biochemical, and structural studies have elucidated the molecular basis for spliceosome catalysis. Splicing is RNA catalyzed and the essential snRNA and protein factors are well-conserved. However, little is known about how non-essential components of the spliceosome contribute to the reaction and modulate the activities of the fundamental core machinery. Ecm2 is a non-essential yeast splicing factor that is a member of the Prp19-related complex of proteins. Cryo-electron microscopy (cryo-EM) structures have revealed that Ecm2 binds the U6 snRNA and is entangled with Cwc2, a factor previously found to promote a catalytically active conformation of the spliceosome. These structures also indicate that Ecm2 and the U2 snRNA likely form a transient interaction during 5' splice site (SS) cleavage. We have characterized genetic interactions between ECM2 and alleles of splicing factors that alter the catalytic steps in splicing. In addition, we have studied how loss of ECM2 impacts splicing of pre-mRNAs containing non-consensus or competing SS. Our results show that ECM2 functions during the catalytic stages of splicing. Our data are consistent with Ecm2 facilitating the formation and stabilization of the 1st-step catalytic site, promoting 2nd-step catalysis, and permiting alternate 5' SS usage. We propose that Cwc2 and Ecm2 can each fine-tune the spliceosome active site in unique ways. Their interaction network may act as a conduit through which splicing of certain pre-mRNAs, such as those containing weak or alternate splice sites, can be regulated.

Impact of cancer-associated mutations in Hsh155/SF3b1 HEAT repeats 9-12 on pre-mRNA splicing in Saccharomyces cerevisiae.

Mutations in the splicing machinery have been implicated in a number of human diseases. Most notably, the U2 small nuclear ribonucleoprotein (snRNP) component SF3b1 has been found to be frequently mutated in blood cancers such as myelodysplastic syndromes (MDS). SF3b1 is a highly conserved HEAT repeat (HR)-containing protein and most of these blood cancer mutations cluster in a hot spot located in HR4-8. Recently, a second mutational hotspot has been identified in SF3b1 located in HR9-12 and is associated with acute myeloid leukemias, bladder urothelial carcinomas, and uterine corpus endometrial carcinomas. The consequences of these mutations on SF3b1 functions during splicing have not yet been tested. We incorporated the corresponding mutations into the yeast homolog of SF3b1 and tested their impact on splicing. We find that all of these HR9-12 mutations can support splicing in yeast, and this suggests that none of them are loss of function alleles in humans. The Hsh155V502F mutation alters splicing of several pre-mRNA reporters containing weak branch sites as well as a genetic interaction with Prp2 and physical interactions with Prp5 and Prp3. The ability of a single allele of Hsh155 to perturb interactions with multiple factors functioning at different stages of the splicing reaction suggests that some SF3b1-mutant disease phenotypes may have a complex origin on the spliceosome.

Dynamics of the DEAD-box ATPase Prp5 RecA-like domains provide a conformational switch during spliceosome assembly.

The DEAD-box family of proteins are ATP-dependent, RNA-binding proteins implicated in many aspects of RNA metabolism. Pre-mRNA splicing in eukaryotes requires three DEAD-box ATPases (Prp5, Prp28 and Sub2), the molecular mechanisms of which are poorly understood. Here, we use single molecule FRET (smFRET) to study the conformational dynamics of yeast Prp5. Prp5 is essential for stable association of the U2 snRNP with the intron branch site (BS) sequence during spliceosome assembly. Our data show that the Prp5 RecA-like domains undergo a large conformational rearrangement only in response to binding of both ATP and RNA. Mutations in Prp5 impact the fidelity of BS recognition and change the conformational dynamics of the RecA-like domains. We propose that BS recognition during spliceosome assembly involves a set of coordinated conformational switches among U2 snRNP components. Spontaneous toggling of Prp5 into a stable, open conformation may be important for its release from U2 and to prevent competition between Prp5 re-binding and subsequent steps in spliceosome assembly.

Functional analysis of Hsh155/SF3b1 interactions with the U2 snRNA/branch site duplex.

SF3b1 is an essential component of the U2 snRNP implicated in branch site (BS) recognition and found to be frequently mutated in several human cancers. While recent structures of yeast and human SF3b1 have revealed its molecular architecture, the importance of specific RNA:protein contacts and conformational changes remains largely uncharacterized. Here, we performed mutational analysis of yeast SF3b1, guided by recent structures of the spliceosome. We find that conserved amino acids contacting the U2 snRNA backbone of the U2/BS duplex are nonessential, and that yeast can tolerate truncation of the HEAT repeats containing these amino acids. The pocket housing the branchpoint adenosine (BP-A) is also amenable to mutation despite strong conservation. However, mutations that support viability can still lead to defects in splicing pre-mRNAs with nonconsensus BS substitutions found at -3, -2, -1, and +1 positions relative to the BP-A or at the branchpoint position. Through the generation of yeast and human chimeric proteins, we further defined the functionally conserved regions of Hsh155 as well as identify changes in BS usage resulting from inclusion of human SF3b1 HEAT repeats. Moreover, these chimeric proteins confer a sensitivity to small molecule inhibition by pladienolide B to yeast splicing. Together, these data reveal the importance of individual contacts of Hsh155/SF3b1 to the U2/BS duplex and define their contribution to BS usage by the spliceosome.

SF3b1 mutations associated with myelodysplastic syndromes alter the fidelity of branchsite selection in yeast.

RNA and protein components of the spliceosome work together to identify the 5΄ splice site, the 3΄ splice site, and the branchsite (BS) of nascent pre-mRNA. SF3b1 plays a key role in recruiting the U2 snRNP to the BS. Mutations in human SF3b1 have been linked to many diseases such as myelodysplasia (MDS) and cancer. We have used SF3b1 mutations associated with MDS to interrogate the role of the yeast ortholog, Hsh155, in BS selection and splicing. These alleles change how the spliceosome recognizes the BS and alter splicing when nonconsensus nucleotides are present at the -2, -1 and +1 positions relative to the branchpoint adenosine. This indicates that changes in BS usage observed in humans with SF3b1 mutations may result from perturbation of a conserved mechanism of BS recognition. Notably, different HSH155 alleles elicit disparate effects on splicing: some increase the fidelity of BS selection while others decrease fidelity. Our data support a model wherein conformational changes in SF3b1 promote U2 association with the BS independently of the action of the DEAD-box ATPase Prp5. We propose that SF3b1 functions to stabilize weak U2/BS duplexes to drive spliceosome assembly and splicing.