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].

Identification of hnRNP C1/C2 as an Autoantigen in Patients with Behcet's Disease.

BACKGROUND: Ribonucleoproteins particles that form the spliceosomes are among the most frequently targeted molecules of the autoimmune response. In the last few years, autoantibodies against all A/B hnRNP proteins have been found in the sera of patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), mixed connective tissue disease (MCTD), and serve as diagnostic markers for several rheumatic diseases. However, the functional role of hnRNP C1/C2 in autoimmune diseases is still not clearly understood. OBJECTIVE: To identify hnRNP C1/C2 as an autoantigen in patients with Behcet's Disease (BD). METHODS: First, HaCaT and EA.hy926 cells were cultured and RNA was extracted. Second, amplification of the corresponding gene by RT-PCR, cloning, and purification techniques was applied to acquire the recombinant protein hnRNP C1/C2. Third, the target protein band was excised from gel electrophoresis, digested with trypsin, and analyzed by (MALDI-TOF/). Finally, Western blotting and ELISA were performed to verify the immunoreactivity of BD serum with recombinant hnRNPC1/C2. RESULTS: Results demonstrated that the reactivity of BD serum against recombinant hnRNP C1/C2 protein was significantly higher as compared to healthy control (P<0.001). CONCLUSION: hnRNP C1/C2 can be considered as a self antigen which might be involved in BD pathology in Hans Chinese population.

A novel nucleocytoplasmic shuttling sequence of DAZAP1, a testis-abundant RNA-binding protein.

Deleted in Azoospermia Associated Protein 1 (DAZAP1) is a ubiquitous RNA-binding protein highly expressed in the human and the mouse testes. It shows a dynamic subcellular localization during spermatogenesis, present predominantly in the nuclei of late-stage spermatocytes and round spermatids and translocated to the cytoplasm during spermatid elongation. To test the hypothesis that DAZAP1 shuttles between the nucleus and the cytoplasm, we studied the nuclear transport of DAZAP1 in somatic cells using immunostaining, heterokaryon formation, and mutagenesis. DAZAP1 is detected exclusively in the nucleus and has the ability to shuttle between the nucleus and the cytoplasm using a highly conserved 25 amino acid segment, designated ZNS, at its C terminus. ZNS shares no sequence homology with other known nuclear localization or export signals. Attachment of ZNS to a red fluorescent protein DsRed2 confers the nucleocytoplasmic shuttling ability to that protein. The nuclear localization of DAZAP1 depends on active transcription. In the presence of an RNA polymerase II inhibitor, DAZAP1 is retained in the cytoplasm. DAZAP1 colocalizes with hnRNP A1 and hnRNP C1 in the nucleus and is a component of the heterogeneous nuclear ribonucleoprotein particles. Our results suggest that DAZAP1 plays a key role in mRNA transport during spermatogenesis.