Aberrant 3′ oligoadenylation of spliceosomal U6 small nuclear RNA in poikiloderma with neutropenia

Abstract

Key Points• Crystal structure of human USB1 identifies it as a member of the LigT-like superfamily of 2H phosphoesterases.• USB1 protects spliceosomal U6 small nuclear RNA from aberrant 3Ј oligoadenylation.The recessive disorder poikiloderma with neutropenia (PN) is caused by mutations in the C16orf57 gene that encodes the highly conserved USB1 protein.Here, we present the 1.1 Å resolution crystal structure of human USB1, defining it as a member of the LigT-like superfamily of 2H phosphoesterases. We show that human USB1 is a distributive 3-5 exoribonuclease that posttranscriptionally removes uridine and adenosine nucleosides from the 3 end of spliceosomal U6 small nuclear RNA (snRNA), directly catalyzing terminal 2, 3 cyclic phosphate formation. USB1 measures the appropriate length of the U6 oligo(U) tail by reading the position of a key adenine nucleotide (A102) and pausing 5 uridine residues downstream. We show that the 3 ends of U6 snRNA in PN patient lymphoblasts are elongated and unexpectedly carry nontemplated 3 oligo(A) tails that are characteristic of nuclear RNA surveillancetargets. Thus, our study reveals a novel quality control pathway in which posttranscriptional 3-end processing by USB1 protects U6 snRNA from targeting and destruction by the nuclear exosome. Our data implicate aberrant oligoadenylation of U6 snRNA in the pathogenesis of the leukemia predisposition disorder PN. (Blood. 2013;121(6):1028-1038)

Incomplete splicing of neutrophil-specific genes affects neutrophil development in a zebrafish model of poikiloderma with neutropenia

“…21 By contrast, USB1 deletion in yeast cells showed lower levels of cellular U6, leading to general pre-mRNA splicing defects. 19 To investigate the effects of usb1 knockdown on the pre-mRNA splicing in zebrafish, we examined the total RNA by sqRT-PCR and analyzed the expression of the genes involving the differentiation and development of neutrophils.…”

“…Despite the loss of USB1 activity, the steady state levels of U6 snRNA were unaffected in PN patients. 21 This might be attributed to the presence of multiple and dispersed U6 snRNA genes that have varied transcriptional efficiencies in humans. 40 Although we have not determined the 3 0 end sequence of U6 snRNA in this study, instead we observed that the expression of U6 snRNA was not altered by the usb1 knockdown in zebrafish.…”

“…19,21 These in vitro and in vivo studies suggest that PN manifestations are not derived from common pre-mRNA splicing defects. Instead, they might have resulted from incomplete splicing of the genes expressed in the tissues that are highly affected by the disease, such as neutrophil precursors.…”

“…In addition, it was demonstrated in vitro that human USB1 is a novel 3 0 -5 0 exoribonuclease that removes terminal oligo(U) and oligo(A) tails at the 3 0 end of U6 snRNA and generates the 3 0 terminal phosphate modification. 21 Deep-sequencing analysis of U6 snRNA from the PN patient lymphoblasts revealed the non-templated addition of more than 2 adenosine nucleotides to the 3 0 end of U6, although no significant difference in the steady-state levels of U6 snRNA was observed. 21 Interestingly, aberrant 3 0 end processing of U6 was observed in the USB1-deficient fission yeasts and the human cells derived from PN patients.…”

“…21 Deep-sequencing analysis of U6 snRNA from the PN patient lymphoblasts revealed the non-templated addition of more than 2 adenosine nucleotides to the 3 0 end of U6, although no significant difference in the steady-state levels of U6 snRNA was observed. 21 Interestingly, aberrant 3 0 end processing of U6 was observed in the USB1-deficient fission yeasts and the human cells derived from PN patients. In human cells, the steadystate levels of U6 were not affected by the diminished USB1 activity, but in yeasts, the cellular U6 levels were greatly reduced, leading to precursor mRNA (pre-mRNA) splicing defects.…”