Biology subjects

Jurica, M. S.

Publications and source records attributed to Jurica, M. S..

8 recordsLinked to original sources

bioRxiv2025

A Yeast-Based High-Throughput Screening Platform for the Discovery of Novel pre-mRNA Splicing Modulators

Pre-mRNA splicing is a crucial process in eukaryotic gene expression, and splicing dysregulation has been linked to various diseases. However, very few small molecules have been discovered that can modulate spliced mRNA formation or inhibit the splicing machinery itself. This study presents a novel high-throughput screening (HTS) platform for identifying compounds that modulate splicing. Our platform comprises a two-tiered screening approach: a primary screen measuring growth inhibition in sensitized in S. cerevisiae (yeast) strains and a secondary screen that relies on production of a fluorescent protein as a readout for splicing inhibition. Using this approach, we identified 4 small molecules that cause accumulation of unspliced pre-mRNA in vivo in yeast. In addition, cancer cells expressing a myelodysplastic syndrome-associated splicing factor mutation (SRSF2P95H) are more sensitive to one of these compounds than those expressing the wild-type version of the protein. Transcriptome analyses showed that this compound causes widespread changes in gene expression in sensitive SRSF2P95H-expressing cells. Our results demonstrate the utility of using a yeast-based HTS to identify compounds capable of changing pre-mRNA splicing outcomes.

biochemistry↗

bioRxiv2025

The human branchpoint-interacting stem loop sequence and structure regulates U2 snRNA expression, branchpoint recognition, and transcriptome.

During pre-mRNA splicing, the branch helix forms when U2 snRNP engages with introns to initiate spliceosome assembly. Its formation is mutually exclusive with the branchpoint-interacting stem loop (BSL) present in U2 snRNA. While BSL structure impacts splicing with the strict consensus branchpoint sequence of yeast introns, its influence in the flexible context of human branchpoints is unknown. We employed an orthogonal U2 snRNA and splicing reporter to examine the impact of perturbing BSL base-pairing and found differential effects on both orthogonal U2 snRNA expression and reporter splicing, indicating that BSL structure influences the biogenesis of U2 snRNP and its function in splicing. Furthermore, high complementarity between the branchpoint sequence and U2 snRNA increases splicing efficiency with wildtype and stabilized BSL, but not when BSL base-pairing is reduced. These data are consistent with complementarity between the intron and the loop of the BSL driving intron-mediated unwinding of the BSL stem. Finally, we investigated transcriptome-wide effects of expressing U2 snRNA with either a cancer-associated BSL mutation or with an altered branchpoint recognition sequence. Similar changes in splicing and gene expression suggests that while altered U2 snRNA is tolerated, cells respond by upregulating genes linked to oncogenic pathways. Main ConclusionsO_LIU2 snRNA BSL base-pairing influences branchpoint sequence recognition and splicing of an orthogonal splicing reporter C_LIO_LIPropose a model for branchpoint sequence complementarity inducing BSL unwinding to promote splicing efficiency C_LIO_LIU2 snRNA BSL and BPRS mutations alter cellular gene expression regulation and splicing C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/673180v2_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@19daa92org.highwire.dtl.DTLVardef@295f40org.highwire.dtl.DTLVardef@2c7f8eorg.highwire.dtl.DTLVardef@16a562f_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

Broad variation in response of individual introns to splicing inhibitors in a humanized yeast strain

Intron branch point (BP) recognition by the U2 snRNP is a critical step of splicing, vulnerable to recurrent cancer mutations and bacterial natural product inhibitors. The BP binds a conserved pocket in the SF3B1 (human) or Hsh155 (yeast) U2 snRNP protein. Amino acids that line this pocket affect binding of splicing inhibitors like Pladienolide-B (Plad-B), such that organisms differ in their sensitivity. To study the mechanism of splicing inhibitor action in a simplified system, we modified the naturally Plad-B resistant yeast Saccharomyces cerevisiae by changing 14 amino acids in the Hsh155 BP pocket to those from human. This humanized yeast grows normally, and splicing is largely unaffected by the mutation. Splicing is inhibited within minutes after addition of Plad-B, and different introns appear inhibited to different extents. Intron-specific inhibition differences are also observed during co-transcriptional splicing in Plad-B using single-molecule intron tracking (SMIT) to minimize gene-specific transcription and decay rates that cloud estimates of inhibition by standard RNA-seq. Comparison of Plad-B intron sensitivities to those of the structurally distinct inhibitor Thailanstatin-A reveals intron-specific differences in sensitivity to different compounds. This work exposes a complex relationship between binding of different members of this class of inhibitors to the spliceosome and intron-specific rates of BP recognition and catalysis. Introns with variant BP sequences seem particularly sensitive, echoing observations from mammalian cells, where monitoring individual introns is complicated by multi-intron gene architecture and alternative splicing. The compact yeast system may hasten characterization of splicing inhibitors, accelerating improvements in selectivity and therapeutic efficacy.

molecular biology↗

bioRxiv2021

A Model for DHX15 Mediated Disassembly of A-Complex Spliceosomes

A critical step of pre-mRNA splicing is the recruitment of U2 snRNP to the branch point sequence of an intron. U2 snRNP conformation changes extensively during branch helix formation and several RNA-dependent-ATPases are implicated in the process. However, the molecular mechanisms involved remain to be fully dissected. We took advantage of the differential nucleotide triphosphates requirements for DExD/H-box enzymes to probe their contributions to in vitro spliceosome assembly. Both ATP and GTP hydrolysis support the formation of A-complex, indicating the activity of a DEAH-enzyme because DEAD-enzymes are selective for ATP. We immunodepleted DHX15 to assess its involvement and although splicing efficiency decreases with reduced DHX15, A-complex accumulation incongruently increases. DHX15 depletion also results in the persistence of the atypical ATP-independent interaction between U2 snRNP and a minimal substrate that is otherwise destabilized in the presence of either ATP or GTP. These results lead us to hypothesize that DHX15 plays a quality control function in U2 snRNPs engagement with an intron. In efforts to identify the RNA target of DHX15, we determined that an extended polypyrimidine tract is not necessary for disruption of the atypical interaction between U2 snRNP and the minimal substrate. We also examined U2 snRNA by RNase H digestion and identified nucleotides in the branch binding region that become accessible with both ATP and GTP hydrolysis, again implicating a DEAH-enzyme. Together, our results demonstrate that multiple ATP-dependent rearrangements are likely involved in U2 snRNP addition to the spliceosome and that DHX15 can have an expanded role in splicing.

molecular biology↗

bioRxiv2021

U2 snRNA structure is influenced by SF3A and SF3B proteins but not by SF3B inhibitors

U2 snRNP is an essential component of the spliceosome. It is responsible for branch point recognition in the spliceosome A-complex via base-pairing of U2 snRNA with an intron to form the branch helix. Small molecule inhibitors target the SF3B component of the U2 snRNP and interfere with A-complex formation during spliceosome assembly. We previously found that the first SF3B inhibited-complex is less stable than A-complex and hypothesized that SF3B inhibitors interfere with U2 snRNA secondary structure changes required to form the branch helix. Using RNA chemical modifiers, we probed U2 snRNA structure in A-complex and SF3B inhibited splicing complexes. The reactivity pattern for U2 snRNA in the SF3B inhibited-complex is indistinguishable from that of A-complex, suggesting that they have the same secondary structure conformation, including the branch helix. This observation suggests SF3B inhibited-complex instability does not stem from an alternate RNA conformation and instead points to the inhibitors interfering with protein component interactions that normally stabilize U2 snRNPs association with an intron. In addition, we probed U2 snRNA in the free U2 snRNP in the presence of SF3B inhibitor and again saw no differences. However, increased protection of nucleotides upstream of Stem I in the absence of SF3A and SF3B proteins suggests a change of secondary structure at the very 5' end of U2 snRNA. Chemical probing of synthetic U2 snRNA in the absence of proteins results in similar protections and predicts a previously uncharacterized extension of Stem I. Because this stem must be disrupted for SF3A and SF3B proteins to stably join the snRNP, the structure has the potential to influence snRNP assembly and recycling after spliceosome disassembly.

molecular biology↗

bioRxiv2020

Identification of herboxidiene structural features that mediate conformation-dependent SF3B1 interactions to inhibit splicing.

Small molecules that target the spliceosome SF3B complex are potent inhibitors of cancer cell growth. The compounds affect an early stage of spliceosome assembly when U2 snRNP first engages the branch point sequence of an intron. Recent cryo-EM models of U2 snRNP before and after intron recognition suggest several large-scale rearrangements of RNA and protein interactions involving SF3B. Employing an inactive herboxidiene analog as a competitor with SF3B inhibitors, we present evidence for multiple conformations of SF3B in the U2 snRNP, only some of which are available for productive inhibitor interactions. We propose that both thermodynamics and an ATP-binding event promote the conformation conducive to SF3B inhibitor interactions. However, SF3B inhibitors do not impact an ATP-dependent rearrangement in U2 snRNP that exposes the branch binding sequence for base pairing. We also report extended structure activity relationship analysis of herboxidiene, which identified features of the tetrahydropyran ring that mediate its interactions with SF3B and its ability to interfere with splicing. In combination with structural models of SF3B interactions with inhibitors, our data leads us to extend the model for early spliceosome assembly and inhibitor mechanism. We postulate that interactions between a carboxylic acid substituent of herboxidiene and positively charged SF3B1 sidechains in the inhibitor binding channel are required to maintain inhibitor occupancy and counteract the SF3B transition to a closed state that is promoted by stable U2 snRNA interactions with the intron.

molecular biology↗

bioRxiv2020

A synthetic small molecule stalls pre-mRNA splicing by promoting an early-stage U2AF2-RNA complex

Dysregulated pre-mRNA splicing is an emerging Achilles heel of cancers and myelodysplasias. To expand the currently limited portfolio of small molecule drug leads, we screened for chemical modulators of the U2AF complex, which nucleates spliceosome assembly and is mutated in myelodysplasias. A hit compound specifically enhances RNA binding by a U2AF2 subunit. Remarkably, the compound inhibits splicing of representative substrates in cells and stalls spliceosome assembly at the stage of U2AF function. Computational docking, together with structure-guided mutagenesis, indicates that the compound bridges an active conformation of the U2AF2 RNA recognition motifs via hydrophobic and electrostatic moieties. Altogether, our results highlight the potential of trapping early spliceosome assembly as an effective pharmacological means to manipulate pre-mRNA splicing. By extension, we suggest that stabilizing inactive checkpoints may offer a breakthrough approach for small molecule inhibition of multi-stage macromolecular assemblies.

biochemistry↗

bioRxiv2018

A two-step probing method to compare lysine accessibility across macromolecular complex conformations

Structural models of multi-megadalton molecular complexes are appearing in increasing numbers, in large part because of technical advances in cryo-electron microscopy realized over the last decade. However, the inherent complexity of large biological assemblies comprising dozens of components often limits the resolution of structural models. Furthermore, multiple functional configurations of a complex can leave a puzzle as to how one intermediate moves to the next stage. Orthogonal biochemical information is crucial to understanding the molecular interactions that drive those rearrangements. We present a two-step method for chemical probing detected by tandem mass-spectrometry to globally assess the reactivity of lysine residues within purified macromolecular complexes. Because lysine side chains often balance the negative charge of RNA in ribonucleoprotein complexes, the method is especially powerful for detecting changes in protein-RNA interactions. Probing the E. coli 30S ribosome subunit showed that the reactivity pattern of lysine residues quantitatively reflects structure models from X-ray crystallography. We assessed differences in two conformations of purified human spliceosomes. Our results demonstrate that this method supplies powerful biochemical information that aids in functional interpretation of atomic models of macromolecular complexes at the intermediate resolution often provided by cryo-electron microscopy.

molecular biology↗