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

Abstract

Biallelic mutations in the untranslated regions (UTRs) of mRNAs are rare causes for monogenetic diseases whose mechanisms remain poorly understood. We investigated a 3 0 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 0 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 0 end processing. Our data suggest that U1 snRNP inhibits cleavage or poly(A) site recognition. This is the first description of a 3 0 UTR mutation that creates a functional 5 0 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.

U1 snRNP-mediated poly(A) site suppression

“…This indicates the presence of a DSE and other auxiliary elements. 16 Most importantly, the ratio between wt and mut mRNA is not affected (Fig. 1G).…”

“…Also, the amount of unprocessed, unspliced RNA at the proximal p14-specific PAS increased by 1.5-fold. 16 Thus, skipping of the natural p14 PAS cannot fully account for the observed reduction in RNA levels ( Fig. 1B and D) and a substantial amount of p14 RNA must be degraded after failure of 3' end processing.…”

“…Interestingly, U1 snRNP-mediated suppression of p14 RNA cleavage/polyadenylation does not affect histone 2A 3' end processing. 16 However, factors such as CPSF and CstF participate in both processes and the cleavage of polyadenylated and histone mRNA is executed by the same endonuclease. 22 Even the beneficial effect of splicing factors such as U2 snRNP can be transferred to histone 3' end processing.…”

“…An RNase protection assay (RPA) and RT-qPCR specific for unprocessed p14 mRNAs displayed more uncleaved p14 transcripts and increased read-through in the case of U1 binding. 16 We have constructed a tandem PAS reporter harboring a duplication of the p14 3'UTR and, thus, two identical PAS. Preliminary data show that the U1 site located in the first 3'UTR is able to partially prevent 3' end formation at the first and second PAS and on top leads to This system can recapitulate the effect of the mutation (Fig.…”

“…16 In our experimental system, we took advantage of minigenes consisting of p14 cDNA, the 3'UTR plus part of the downstream genomic region (Fig. 1A).…”