Correction: Pathological mechanism and antisense oligonucleotide-mediated rescue of a non-coding variant suppressing factor 9 RNA biogenesis leading to hemophilia B.

[This corrects the article DOI: 10.1371/journal.pgen.1008690.].

Pathological mechanism and antisense oligonucleotide-mediated rescue of a non-coding variant suppressing factor 9 RNA biogenesis leading to hemophilia B.

Loss-of-function mutations in the human coagulation factor 9 (F9) gene lead to hemophilia B. Here, we dissected the consequences and the pathomechanism of a non-coding mutation (c.2545A>G) in the F9 3' untranslated region. Using wild type and mutant factor IX (FIX) minigenes we revealed that the mutation leads to reduced F9 mRNA and FIX protein levels and to lower coagulation activity of cell culture supernatants. The phenotype could not be compensated by increased transcription. The pathomechanism comprises the de novo creation of a binding site for the spliceosomal component U1snRNP, which is able to suppress the nearby F9 poly(A) site. This second, splicing-independent function of U1snRNP was discovered previously and blockade of U1snRNP restored mutant F9 mRNA expression. In addition, we explored the vice versa approach and masked the mutation by antisense oligonucleotides resulting in significantly increased F9 mRNA expression and coagulation activity. This treatment may transform the moderate/severe hemophilia B into a mild or subclinical form in the patients. This antisense based strategy is applicable to other mutations in untranslated regions creating deleterious binding sites for cellular proteins.

Very early onset inflammatory bowel disease associated with aberrant trafficking of IL-10R1 and cure by T cell replete haploidentical bone marrow transplantation.

PURPOSE: Loss-of-function mutations in IL10 and IL10R cause very early onset inflammatory bowel disease (VEO-IBD). Here, we investigated the molecular pathomechanism of a novel intronic IL10RA mutation and describe a new therapeutic approach of T cell replete haploidentical hematopoietic stem cell transplantation (HSCT). METHODS: Clinical data were collected by chart review. Genotypes of IL10 and IL10R genes were determined by Sanger sequencing. Expression and function of mutated IL-10R1 were assessed by quantitative PCR, Western blot analysis, enzyme-linked immunosorbent assays, confocal microscopy, and flow cytometry. RESULTS: We identified a novel homozygous point mutation in intron 3 of the IL10RA (c.368-10C > G) in three related children with VEO-IBD. Bioinformatical analysis predicted an additional 3' splice site created by the mutation. Quantitative PCR analysis showed normal mRNA expression of mutated IL10RA. Sequencing of the patient's cDNA revealed an insertion of the last nine nucleotides of intron 3 as a result of aberrant splicing. Structure-based modeling suggested misfolding of mutated IL-10R1. Western blot analysis demonstrated a different N-linked glycosylation pattern of mutated protein. Immunofluorescence and FACS analysis revealed impaired expression of mutated IL-10R1 at the plasma membrane. In the absence of HLA-identical donors, T cell replete haploidentical HSCT was successfully performed in two patients. CONCLUSIONS: Our findings expand the spectrum of IL10R mutations in VEO-IBD and emphasize the need for genetic diagnosis of mutations in conserved non-coding sequences of candidate genes. Transplantation of haploidentical stem cells represents a curative therapy in IL-10R-deficient patients, but may be complicated by non-engraftment.

U1snRNP-mediated suppression of polyadenylation in conjunction with the RNA structure controls poly (A) site selection in foamy viruses.

BACKGROUND: During reverse transcription, retroviruses duplicate the long terminal repeats (LTRs). These identical LTRs carry both promoter regions and functional polyadenylation sites. To express full-length transcripts, retroviruses have to suppress polyadenylation in the 5'LTR and activate polyadenylation in the 3'LTR. Foamy viruses have a unique LTR structure with respect to the location of the major splice donor (MSD), which is located upstream of the polyadenylation signal. RESULTS: Here, we describe the mechanisms of foamy viruses regulating polyadenylation. We show that binding of the U1 small nuclear ribonucleoprotein (U1snRNP) to the MSD suppresses polyadenylation at the 5'LTR. In contrast, polyadenylation at the 3'LTR is achieved by adoption of a different RNA structure at the MSD region, which blocks U1snRNP binding and furthers RNA cleavage and subsequent polyadenylation. CONCLUSION: Recently, it was shown that U1snRNP is able to suppress the usage of intronic cryptic polyadenylation sites in the cellular genome. Foamy viruses take advantage of this surveillance mechanism to suppress premature polyadenylation at the 5'end of their RNA. At the 3'end, Foamy viruses use a secondary structure to presumably block access of U1snRNP and thereby activate polyadenylation at the end of the genome. Our data reveal a contribution of U1snRNP to cellular polyadenylation site selection and to the regulation of gene expression.

Critical role for miR-181a/b-1 in agonist selection of invariant natural killer T cells.

T-cell receptor (TCR) signal strength determines selection and lineage fate at the CD4(+)CD8(+) double-positive stage of intrathymic T-cell development. Members of the miR-181 family constitute the most abundantly expressed microRNA at this stage of T-cell development. Here we show that deletion of miR-181a/b-1 reduced the responsiveness of double-positive thymocytes to TCR signals and virtually abrogated early invariant natural killer T (iNKT) cell development, resulting in a dramatic reduction in iNKT cell numbers in thymus as well as in the periphery. Increased concentrations of agonist ligand rescued iNKT cell development in miR-181a/b-1(-/-) mice. Our results define a critical role of miR-181a/b-1 in early iNKT cell development and show that miR-181a/b-1 sets a TCR signaling threshold for agonist selection.

U1 snRNP-mediated poly(A) site suppression: beneficial and deleterious for mRNA fate.

The spliceosomal component U1snRNP commits pre-mRNAs to the splicing pathway. Recently, a nuclear RNA surveillance function has been ascribed to U1, namely the suppression of intronic polyadenylation sites. This surveillance holds regulatory potential as it alters the 3' ends of certain receptor tyrosine kinase mRNAs. However, suppression of 3' end processing by U1 snRNP is also the cause of a severe genetic disorder. We described a 3'UTR point mutation creating a 5'SS leading to U1-mediated suppression of 3' end formation. Thus, the inhibitory function of U1 is both beneficial and deleterious where misled. The exact mechanism of how U1 interferes with 3' end processing remains unclear. According to our data, U1 snRNP already interferes with cleavage or poly(A) site selection instead of directly inhibiting poly(A) polymerase as previously assumed. Here, we present alternative models for U1-mediated poly(A) site suppression and discuss the implications for RNA quality control and disease-related mutations.

A complex immunodeficiency is based on U1 snRNP-mediated poly(A) site suppression.

Biallelic mutations in the untranslated regions (UTRs) of mRNAs are rare causes for monogenetic diseases whose mechanisms remain poorly understood. We investigated a 3'UTR mutation resulting in a complex immunodeficiency syndrome caused by decreased mRNA levels of p14/robld3 by a previously unknown mechanism. Here, we show that the mutation creates a functional 5' splice site (SS) and that its recognition by the spliceosomal component U1 snRNP causes p14 mRNA suppression in the absence of splicing. Histone processing signals are able to rescue p14 expression. Therefore, the mutation interferes only with canonical poly(A)-site 3' end processing. Our data suggest that U1 snRNP inhibits cleavage or poly(A) site recognition. This is the first description of a 3'UTR mutation that creates a functional 5'SS causative of a monogenetic disease. Moreover, our data endorse the recently described role of U1 snRNP in suppression of intronic poly(A) sites, which is here deleterious for p14 mRNA biogenesis.