Minor Spliceosomal 65K/RNPC3 Interacts with ANKRD11 and Mediates HDAC3-Regulated Histone Deacetylation and Transcription.

RNA splicing is crucial in the multilayer regulatory networks for gene expression, making functional interactions with DNA- and other RNA-processing machineries in the nucleus. However, these established couplings are all major spliceosome-related; whether the minor spliceosome is involved remains unclear. Here, through affinity purification using Drosophila lysates, an interaction is identified between the minor spliceosomal 65K/RNPC3 and ANKRD11, a cofactor of histone deacetylase 3 (HDAC3). Using a CRISPR/Cas9 system, Deletion strains are constructed and found that both Dm65KΔ/Δ and Dmankrd11Δ/Δ mutants have reduced histone deacetylation at Lys9 of histone H3 (H3K9) and Lys5 of histone H4 (H4K5) in their heads, exhibiting various neural-related defects. The 65K-ANKRD11 interaction is also conserved in human cells, and the HsANKRD11 middle-uncharacterized domain mediates Hs65K association with HDAC3. Cleavage under targets and tagmentation (CUT&Tag) assays revealed that HsANKRD11 is a bridging factor, which facilitates the synergistic common chromatin-binding of HDAC3 and Hs65K. Knockdown (KD) of HsANKRD11 simultaneously decreased their common binding, resulting in reduced deacetylation of nearby H3K9. Ultimately, this study demonstrates that expression changes of many genes caused by HsANKRD11-KD are due to the decreased common chromatin-binding of HDAC3 and Hs65K and subsequently reduced deacetylation of H3K9, illustrating a novel and conserved coupling mechanism that links the histone deacetylation with minor spliceosome for the regulation of gene expression.

Anti-U11/U12 Antibodies as a Rare but Important Biomarker in Patients with Systemic Sclerosis: A Narrative Review.

Anti-nuclear (ANA) are present in approximately 90% of systemic sclerosis (SSc) patients and are key biomarkers in supporting the diagnosis and determining the prognosis of this disease. In addition to the classification criteria autoantibodies for SSc [i.e., anti-centromere, anti-topoisomerase I (Scl-70), anti-RNA polymerase III], other autoantibodies have been associated with important SSc phenotypes. Among them, anti-U11/U12 ribonucleoprotein (RNP) antibodies, also known as anti-RNPC-3, were first reported in a patient with SSc, but very little is known about their association and clinical utility. The U11/U12 RNP macromolecular complex consists of several proteins involved in alternative mRNA splicing. More recent studies demonstrated associations of anti-anti-U11/U12 antibodies with SSc and severe pulmonary fibrosis as well as with moderate to severe gastrointestinal dysmotility. Lastly, anti-U11/U12 autoantibodies have been strongly associated with malignancy in SSc patients. Here, we aimed to summarize the knowledge of anti-U11/U12/RNPC-3 antibodies in SSc, including their seroclinical associations in a narrative literature review.

Anti-RNPC-3 antibody predicts poor prognosis in patients with interstitial lung disease associated to systemic sclerosis.

OBJECTIVE: To analyse the prevalence, the clinical characteristics, the overall survival and the event-free survival (EFS) of SSc patients who express anti-U11/U12 RNP (RNPC-3) antibodies. METHODS: A total of 447 SSc patients from Barcelona (n = 286) and Milan (n = 161) were selected. All samples were tested using a particle-based multi-analyte technology. We compared anti-RNPC-3 positive and negative patients. Epidemiological, clinical features and survival were analysed. End-stage lung disease (ESLD) was defined if the patient developed forced vital capacity <50% of predicted, needed oxygen therapy or lung transplantation. EFS was defined as the period of time free of either ESLD or death. RESULTS: Nineteen of 447 (4.3%) patients had anti-RNPC-3 antibodies and interstitial lung disease (ILD) was more frequent (11, 57.9% vs 144, 33.6%, P =0.030) in individuals with anti-RNPC-3 antibodies. More patients reached ESLD in the positive group (7, 36.8% vs 74, 17.3%, P = 0.006), and a higher use of non-glucocorticoid immunosuppressive drugs was observed (11, 57.9% vs 130, 30.4%, P = 0.012). Anti-RNPC-3 positive patients had lower EFS, both in the total cohort (log-rank P =0.001), as well as in patients with ILD (log-rank P = 0.002). In multivariate Cox regression analysis, diffuse cutaneous subtype, age at onset, the presence of ILD or pulmonary arterial hypertension and the expression of anti-RNPC-3 positivity or anti-topo I were independently associated with worse EFS. CONCLUSION: The presence of anti-RNPC-3 was associated with higher frequency of ILD and either ESLD or death. These data suggest anti-RNPC-3 is an independent poor prognosis antibody in SSc, especially if ILD is also present.

Mutations in the U11/U12-65K protein associated with isolated growth hormone deficiency lead to structural destabilization and impaired binding of U12 snRNA.

Mutations in the components of the minor spliceosome underlie several human diseases. A subset of patients with isolated growth hormone deficiency (IGHD) harbors mutations in the RNPC3 gene, which encodes the minor spliceosome-specific U11/U12-65K protein. Although a previous study showed that IGHD patient cells have defects in U12-type intron recognition, the biochemical effects of these mutations on the 65K protein have not been characterized. Here, we show that a proline-to-threonine missense mutation (P474T) and a nonsense mutation (R502X) in the C-terminal RNA recognition motif (C-RRM) of the 65K protein impair the binding of 65K to U12 and U6atac snRNAs. We further show that the nonsense allele is targeted to the nonsense-mediated decay (NMD) pathway, but in an isoform-specific manner, with the nuclear-retained 65K long-3'UTR isoform escaping the NMD pathway. In contrast, the missense P474T mutation leads, in addition to the RNA-binding defect, to a partial defect in the folding of the C-RRM and reduced stability of the full-length protein, thus reducing the formation of U11/U12 di-snRNP complexes. We propose that both the C-RRM folding defect and NMD-mediated decrease in the levels of the U11/U12-65K protein reduce formation of the U12-type intron recognition complex and missplicing of a subset of minor introns leading to pituitary hypoplasia and a subsequent defect in growth hormone secretion.

Structural and mechanistic insights into human splicing factor SF3b complex derived using an integrated approach guided by the cryo-EM density maps.

Pre-mRNA splicing in eukaryotes is performed by the spliceosome, a highly complex macromolecular machine. SF3b is a multi-protein complex which recognizes the branch point adenosine of pre-mRNA as part of a larger U2 snRNP or U11/U12 di-snRNP in the dynamic spliceosome machinery. Although a cryo-EM map is available for human SF3b complex, the structure and relative spatial arrangement of all components in the complex are not yet known. We have recognized folds of domains in various proteins in the assembly and generated comparative models. Using an integrative approach involving structural and other experimental data, guided by the available cryo-EM density map, we deciphered a pseudo-atomic model of the closed form of SF3b which is found to be a "fuzzy complex" with highly flexible components and multiplicity of folds. Further, the model provides structural information for 5 proteins (SF3b10, SF3b155, SF3b145, SF3b130 and SF3b14b) and localization information for 4 proteins (SF3b10, SF3b145, SF3b130 and SF3b14b) in the assembly for the first time. Integration of this model with the available U11/U12 di-snRNP cryo-EM map enabled elucidation of an open form. This now provides new insights on the mechanistic features involved in the transition between closed and open forms pivoted by a hinge region in the SF3b155 protein that also harbors cancer causing mutations. Moreover, the open form guided model of the 5' end of U12 snRNA, which includes the branch point duplex, shows that the architecture of SF3b acts as a scaffold for U12 snRNA: pre-mRNA branch point duplex formation with potential implications for branch point adenosine recognition fidelity.

Structural features important for the U12 snRNA binding and minor spliceosome assembly of Arabidopsis U11/U12-small nuclear ribonucleoproteins.

Although seven proteins unique to U12 intron-specific minor spliceosomes, denoted as U11/U12-65K, -59K, -48K, -35K, -31K, -25K, and -20K, have been identified in humans and the roles of some of them have been demonstrated, the functional role of most of these proteins in plants is not understood. A recent study demonstrated that Arabidopsis U11/U12-65K is essential for U12 intron splicing and normal plant development. However, the structural features and sequence motifs important for 65 K binding to U12 snRNA and other spliceosomal proteins remain unclear. Here, we demonstrated by domain-deletion analysis that the C-terminal region of the 65 K protein bound specifically to the stem-loop III of U12 snRNA, whereas the N-terminal region of the 65 K protein was responsible for interacting with the 59 K protein. Analysis of the interactions between each snRNP protein using yeast two-hybrid analysis and in planta bimolecular fluorescence complementation and luciferase complementation imaging assays demonstrated that the core interactions among the 65 K, 59 K, and 48 K proteins were conserved between plants and animals, and multiple interactions were observed among the U11/U12-snRNP proteins. Taken together, these results reveal that U11/U12-65K is an indispensible component of the minor spliceosome complex by binding to both U11/U12-59K and U12 snRNA, and that multiple interactions among the U11/U12-snRNP proteins are necessary for minor spliceosome assembly.

The Arabidopsis U11/U12-65K is an indispensible component of minor spliceosome and plays a crucial role in U12 intron splicing and plant development.

The U12-dependent introns have been identified in a wide range of eukaryotes and are removed from precursor-mRNAs by U12 intron-specific minor spliceosome. Although several proteins unique to minor spliceosome have been identified, the nature of their effect on U12 intron splicing as well as plant growth and development remain largely unknown. Here, we characterized the functional role of an U12-type spliceosomal protein, U11/U12-65K in Arabidopsis thaliana. The transgenic knockdown plants generated by artificial miRNA-mediated silencing strategy exhibited severe defect in growth and development, such as severely arrested primary inflorescence stems, serrated leaves, and the formation of many rosette leaves after bolting. RNA sequencing and reverse transcription polymerase chain reaction (RT-PCR) analyses revealed that splicing of 198 out of the 234 previously predicted U12 intron-containing genes and 32 previously unidentified U12 introns was impaired in u11/u12-65k mutant. Moreover, the U11/U12-65K mutation affected alternative splicing, as well as U12 intron splicing, of many introns. Microarray analysis revealed that the genes involved in cell wall biogenesis and function, plant development, and metabolic processes are differentially expressed in the mutant plants. U11/U12-65K protein bound specifically to U12 small nuclear RNA (snRNA), which is necessary for branch-point site recognition. Taken together, these results provide clear evidence that U11/U12-65K is an indispensible component of minor spliceosome and involved in U12 intron splicing and alternative splicing of many introns, which is crucial for plant development.