Identification of HnRNPC as a novel Tau exon 10 splicing factor using RNA antisense purification mass spectrometry.

Alternative splicing in Tau exon 10 generates 3 R- and 4 R-Tau proteoforms, which have equal abundance in healthy adult human brain. Aberrant alternative splicing in Tau exon 10 leads to distortion of the balanced 3 R- and 4 R-Tau expression levels, which is a causal factor to trigger toxic Tau aggregation, neuron dysfunction and patient death in a group of neurodegenerative diseases known as tauopathies. Hence, identification of regulators upstream of the Tau exon 10 splicing events are crucial to understanding pathogenic mechanisms driving tauopathies. In this study, we used RNA Antisense Purification with Mass Spectrometry (RAP-MS) analysis to identify RNA-binding proteins (RBPs) that interact with the Tau pre-mRNA near exon 10. Among the newly identified RBP candidates, we show that knockdown of hnRNPC induces Tau exon 10 skipping whereas overexpression of hnRNPC promotes Tau exon 10 inclusion. In addition, we show that hnRNPC interacts with the poly-uridine (U-tract) sequences in introns 9 and 10 of Tau pre-mRNA. Mutation of these U-tract motifs abolished binding of hnRNPC with Tau pre-mRNA fragment and blocked its impact on Tau exon 10 inclusion. These findings indicate that hnRNPC binds and utilizes these U-tract motifs located in introns 9 and 10 of Tau pre-mRNA to promote Tau exon 10 inclusion. Intriguingly, high hnRNPC expression level is associated with progressive supranuclear palsy (PSP), a sporadic tauopathy with pathological accumulation of Tau species that contain exon 10, which suggests a putative therapeutic role of hnRNPC for PSP treatment. [Figure: see text].

Antibody-based biosensor to detect oncogenic splicing factor Sam68 for the diagnosis of lung cancer.

OBJECTIVE: The present work aimed to investigate the potential utility of Sam68 protein as a prognostic marker in lung cancer. Then an electrochemical immunosensor is fabricated that is sufficiently sensitive to detect Sam68. RESULTS: Analysis of stage-specific Lung cancer microarray data shows that differential expression of Sam68 is associated with cancer stage and monotonically increases from early tumor stage to advanced metastatic stage. Moreover, the higher expression of Sam68 results in reduced survival of lung cancer patients. Based on these observations, an electrochemical immunosensor was developed for the quantification of Sam68 protein. The target protein was captured by the Anti-Sam68 antibody that was immobilized on the modified Glassy carbon electrode. The stepwise assembly process was characterized by cyclic voltammetry and electrochemical impedance spectroscopy. This fabricated immunosensor displayed good analytical performance in comparison to commercial ELISA kit with good sensitivity, lower detection limit (LOD) of 10.5 pg mL-1, and wide linear detection range from 1 to 5 µg mL-1. This method was validated with satisfactory detection of Sam68 protein in lung adenocarcinoma cell line, NCI-H23. Besides, spike and recovery assay reconfirm that the sensor can precisely quantify Sam68 protein in a complex physiological sample. CONCLUSION: We conclude Sam68 as a valuable prognostic biomarker for early detection of lung cancer. Moreover, we report the first study on the development of an electrochemical immunosensor for the detection of Sam68. The fabricated immunosensor exhibit excellent analytical performance, which can accurately predict the lung cancer patient pathological state.

Flexible Binding of m6A Reader Protein YTHDC1 to Its Preferred RNA Motif.

N6-Methyladenosine (m6A) is the most prevalent chemical modification in human mRNAs. Its recognition by reader proteins enables many cellular functions, including splicing and translation of mRNAs. However, the binding mechanisms of m6A-containing RNAs to their readers are still elusive due to the unclear roles of m6A-flanking ribonucleotides. Here, we use a model system, YTHDC1 with its RNA motif 5'-G-2G-1(m6A)C+1U+2-3', to investigate the binding mechanisms by atomistic simulations, X-ray crystallography, and isothermal titration calorimetry. The experimental data and simulation results show that m6A is captured by an aromatic cage of YTHDC1 and the 3' terminus nucleotides are stabilized by cation-π-π interactions, while the 5' terminus remains flexible. Notably, simulations of unbound RNA motifs reveal that the methyl group of m6A and the 5' terminus shift the conformational preferences of the oligoribonucleotide to the bound-like conformation, thereby facilitating the association process. The binding mechanisms may help in the discovery of chemical probes against m6A reader proteins.

Identification of proteins associated with splicing factors Ntr1, Ntr2, Brr2 and Gpl1 in the fission yeast Schizosaccharomyces pombe.

The spliceosome is a complex molecular machine assembled from many components, which catalyzes the removal of introns from mRNA precursors. Our previous study revealed that the Nrl1 (NRDE-2 like 1) protein associates with spliceosome proteins and regulates pre-mRNA splicing and homologous recombination-dependent R-loop formation in the fission yeast Schizosaccharomyces pombe. Here, we identify proteins associated with splicing factors Ntr1, Ntr2, Brr2 and Gpl1, a poorly characterized G-patch domain-containing protein required for efficient splicing. This work provides new evidence that Nrl1 and splicing factors physically interact and reveals additional insights into the protein interaction network of the spliceosome. We discuss implications of these findings in the light of recent progress in our understanding of how Nrl1 and splicing factors ensure genome stability.

The cryo-EM structure of the SF3b spliceosome complex bound to a splicing modulator reveals a pre-mRNA substrate competitive mechanism of action.

Somatic mutations in spliceosome proteins lead to dysregulated RNA splicing and are observed in a variety of cancers. These genetic aberrations may offer a potential intervention point for targeted therapeutics. SF3B1, part of the U2 small nuclear RNP (snRNP), is targeted by splicing modulators, including E7107, the first to enter clinical trials, and, more recently, H3B-8800. Modulating splicing represents a first-in-class opportunity in drug discovery, and elucidating the structural basis for the mode of action opens up new possibilities for structure-based drug design. Here, we present the cryogenic electron microscopy (cryo-EM) structure of the SF3b subcomplex (SF3B1, SF3B3, PHF5A, and SF3B5) bound to E7107 at 3.95 Å. This structure shows that E7107 binds in the branch point adenosine-binding pocket, forming close contacts with key residues that confer resistance upon mutation: SF3B1R1074H and PHF5AY36C The structure suggests a model in which splicing modulators interfere with branch point adenosine recognition and supports a substrate competitive mechanism of action (MOA). Using several related chemical probes, we validate the pose of the compound and support their substrate competitive MOA by comparing their activity against both strong and weak pre-mRNA substrates. Finally, we present functional data and structure-activity relationship (SAR) on the PHF5AR38C mutation that sensitizes cells to some chemical probes but not others. Developing small molecule splicing modulators represents a promising therapeutic approach for a variety of diseases, and this work provides a significant step in enabling structure-based drug design for these elaborate natural products. Importantly, this work also demonstrates that the utilization of cryo-EM in drug discovery is coming of age.

Proteomic analysis of polyribosomes identifies splicing factors as potential regulators of translation during mitosis.

Precise regulation of mRNA translation is critical for proper cell division, but little is known about the factors that mediate it. To identify mRNA-binding proteins that regulate translation during mitosis, we analyzed the composition of polysomes from interphase and mitotic cells using unbiased quantitative mass-spectrometry (LC-MS/MS). We found that mitotic polysomes are enriched with a subset of proteins involved in RNA processing, including alternative splicing and RNA export. To demonstrate that these may indeed be regulators of translation, we focused on heterogeneous nuclear ribonucleoprotein C (hnRNP C) as a test case and confirmed that it is recruited to elongating ribosomes during mitosis. Then, using a combination of pulsed SILAC, metabolic labeling and ribosome profiling, we showed that knockdown of hnRNP C affects both global and transcript-specific translation rates and found that hnRNP C is specifically important for translation of mRNAs that encode ribosomal proteins and translation factors. Taken together, our results demonstrate how proteomic analysis of polysomes can provide insight into translation regulation under various cellular conditions of interest and suggest that hnRNP C facilitates production of translation machinery components during mitosis to provide daughter cells with the ability to efficiently synthesize proteins as they enter G1 phase.