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

Abstract

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 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 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.

Early developmental arrest and impaired gastrointestinal homeostasis in U12-dependent splicing-defective Rnpc3-deficient mice

“…In the single family affected, the RNPC3 mutations occurred in the part of the gene encoding the more C-terminal of its two RNA binding domains. This has been shown to disrupt binding of the protein to the U12 and U6atac snRNAs in the U11/U12 di-snRNP intron recognition complex (Norppa et al 2018). One sequence variant causes an amino acid change at codon 474 (P474T) and the second introduces a stop codon at position 502 (R502X) of the 517 aa/65kDa protein, resulting in impaired expression of the corresponding mRNA (Argente et al 2014).…”

“…In our zebrafish study, the digestive organs and several other tissues that exhibit high rates of proliferation during larval development were the most severely affected by 65K loss. In contrast, the inheritance of two different RNPC3 alleles in human revealed an important but discrete role for 65K in pituitary gland development (Argente et al 2014;Norppa et al 2018), which could be managed effectively by administration of growth hormone. These dissimilar outcomes from 65K deficiency during zebrafish and human development suggest the presence of tissue-specific contexts during development when critical thresholds of minor class splicing efficiency must be met.…”

An Integrated Model of Minor Intron Emergence and Conservation

“…In human, mutation in BRAF results in multiple types of cancers, as well as Noonan syndrome and Costello syndrome, both of which share characteristics of short stature, skeletal abnormalities, craniofacial defects, and heart malfunction (Davies et al, 2002; Tartaglia et al, 2011). Moreover, mutation in RNPC3 , which encodes the minor spliceosome-specific U11/U12-65K protein, results in isolated growth hormone deficiency (IGHD) (Argente et al, 2014; Norppa et al, 2018). Patients with IGHD have short stature due to pituitary hypoplasia and thus reduced growth hormone production (Argente et al, 2014).…”

Response to growth hormone in patients with RNPC 3 mutations

“…RNPC3 encodes a 65‐kDa protein that is a structural component of the U11/U12 small nuclear ribonucleoprotein of the minor spliceosome (Verma et al , ). Mutations in RNPC3 lead to structural destabilization of the 65‐kDa protein, impaired binding of U12 snRNA, and global defects in splicing of U12‐type introns (Argente et al , ; Norppa et al , ).…”