Characterization of Cwc2, U6 snRNA, and Prp8 interactions destabilized by Prp16 ATPase at the transition between the first and second steps of splicing.

The spliceosome performs two consecutive transesterification reactions using one catalytic center, thus requiring its rearrangement between the two catalytic steps of splicing. The Prp16 ATPase facilitates exit from the first-step conformation of the catalytic center by destabilizing some interactions important for catalysis. To better understand rearrangements within the Saccharomyces cerevisiae catalytic center, we characterize factors that modulate the function of Prp16: Cwc2, N-terminal domain of Prp8, and U6-41AACAAU46 region. Alleles of these factors were identified through genetic screens for mutants that correct cs defects of prp16-302 alleles. Several of the identified U6, cwc2, and prp8 alleles are located in close proximity of each other in cryo-EM structures of the spliceosomal catalytic conformations. Cwc2 and U6 interact with the intron sequences in the first step, but they do not seem to contribute to the stability of the second-step catalytic center. On the other hand, the N-terminal segment of Prp8 not only affects intron positioning for the first step, but it also makes important contacts in the proximity of the active site for both the first and second steps of splicing. By identifying interactions important for the stability of catalytic conformations, our genetic analyses indirectly inform us about features of the transition-state conformation of the spliceosome.

U4 snRNP inhibits premature cleavage and polyadenylation of pre-mRNAs.

The essential role of U4 snRNP in pre-messenger RNA (mRNA) splicing has been well established. In this study, we utilized an antisense morpholino oligonucleotide (AMO) specifically targeting U4 snRNA to achieve functional knockdown of U4 snRNP in HeLa cells. Our results showed that this knockdown resulted in global intronic premature cleavage and polyadenylation (PCPA) events, comparable to the effects observed with U1 AMO treatment, as demonstrated by mRNA 3'-seq analysis. Furthermore, our study suggested that this may be a common phenomenon in both human and mouse cell lines. Additionally, we showed that U4 AMO treatment disrupted transcription elongation, as evidenced by chromatin immunoprecipitation sequencing (ChIP-seq) analysis for RNAPII. Collectively, our results identified a unique role for U4 snRNP in the inhibition of PCPA and indicated a model wherein splicing intrinsically inhibits intronic cleavage and polyadenylation in the context of cotranscriptional mRNA processing.

Tracing Allostery in the Spliceosome Ski2-like RNA Helicase Brr2.

RNA ATPases/helicases remodel substrate RNA-protein complexes in distinct ways. The different RNA ATPases/helicases, taking part in the spliceosome complex, reshape the RNA/RNA-protein contacts to enable premature-mRNA splicing. Among them, the bad response to refrigeration 2 (Brr2) helicase promotes U4/U6 small nuclear (sn)RNA unwinding via ATP-driven translocation of the U4 snRNA strand, thus playing a pivotal role during the activation, catalytic, and disassembly phases of splicing. The plastic Brr2 architecture consists of an enzymatically active N-terminal cassette (N-cassette) and a structurally similar but inactive C-terminal cassette (C-cassette). The C-cassette, along with other allosteric effectors and regulators, tightly and timely controls Brr2's function via an elusive mechanism. Here, microsecond-long molecular dynamics simulations, dynamical network theory, and community network analysis are combined to elucidate how allosteric effectors/regulators modulate the Brr2 function. We unexpectedly reveal that U4 snRNA itself acts as an allosteric regulator, amplifying the cross-talk of distal Brr2 domains and triggering a conformational reorganization of the protein. Our findings offer fundamental understanding into Brr2's mechanism of action and broaden our knowledge on the sophisticated regulatory mechanisms by which spliceosome ATPases/helicases control gene expression. This includes their allosteric regulation exerted by client RNA strands, a mechanism that may be broadly applicable to other RNA-dependent ATPases/helicases.

Xrp1 governs the stress response program to spliceosome dysfunction.

Co-transcriptional processing of nascent pre-mRNAs by the spliceosome is vital to regulating gene expression and maintaining genome integrity. Here, we show that the deficiency of functional U5 small nuclear ribonucleoprotein particles (snRNPs) in Drosophila imaginal cells causes extensive transcriptome remodeling and accumulation of highly mutagenic R-loops, triggering a robust stress response and cell cycle arrest. Despite compromised proliferative capacity, the U5 snRNP-deficient cells increased protein translation and cell size, causing intra-organ growth disbalance before being gradually eliminated via apoptosis. We identify the Xrp1-Irbp18 heterodimer as the primary driver of transcriptional and cellular stress program downstream of U5 snRNP malfunction. Knockdown of Xrp1 or Irbp18 in U5 snRNP-deficient cells attenuated JNK and p53 activity, restored normal cell cycle progression and growth, and inhibited cell death. Reducing Xrp1-Irbp18, however, did not rescue the splicing defects, highlighting the requirement of accurate splicing for cellular and tissue homeostasis. Our work provides novel insights into the crosstalk between splicing and the DNA damage response and defines the Xrp1-Irbp18 heterodimer as a critical sensor of spliceosome malfunction and mediator of the stress-induced cellular senescence program.

The removal of introns and the joining of exons into mature mRNA by the spliceosome is crucial in regulating gene expression, simultaneously safeguarding genome integrity and enhancing proteome diversity in multicellular organisms. Spliceosome dysfunction is thus associated with various diseases and organismal aging. Our study describes the cascade of events in response to spliceosome dysfunction. We identified two transcription factors as drivers of a stress response program triggered by spliceosome dysfunction, which dramatically remodel gene expression to protect tissue integrity and induce a senescent-like state in damaged cells prior to their inevitable elimination. Together, we highlight the indispensable role of spliceosomes in maintaining homeostasis and implicate spliceosome dysfunction in senescent cell accumulation associated with the pathomechanisms of spliceopathies and aging.

Pathogenic Variants in USH1G/SANS Alter Protein Interaction with Pre-RNA Processing Factors PRPF6 and PRPF31 of the Spliceosome.

Pre-mRNA splicing is an essential process orchestrated by the spliceosome, a dynamic complex assembled stepwise on pre-mRNA. We have previously identified that USH1G protein SANS regulates pre-mRNA splicing by mediating the intranuclear transfer of the spliceosomal U4/U6.U5 tri-snRNP complex. During this process, SANS interacts with the U4/U6 and U5 snRNP-specific proteins PRPF31 and PRPF6 and regulates splicing, which is disturbed by variants of USH1G/SANS causative for human Usher syndrome (USH), the most common form of hereditary deaf-blindness. Here, we aim to gain further insights into the molecular interaction of the splicing molecules PRPF31 and PRPF6 to the CENTn domain of SANS using fluorescence resonance energy transfer assays in cells and in silico deep learning-based protein structure predictions. This demonstrates that SANS directly binds via two distinct conserved regions of its CENTn to the two PRPFs. In addition, we provide evidence that these interactions occur sequentially and a conformational change of an intrinsically disordered region to a short α-helix of SANS CENTn2 is triggered by the binding of PRPF6. Furthermore, we find that pathogenic variants of USH1G/SANS perturb the binding of SANS to both PRPFs, implying a significance for the USH1G pathophysiology.