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Myeloid malignancies with DDX41 mutations are often associated with bone marrow failure and cytopenia before overt disease manifestation. However, the mechanisms underlying these specific conditions remain elusive. Here, we demonstrate that loss of DDX41 function impairs efficient RNA splicing, resulting in DNA replication stress with excess R-loop formation. Mechanistically, DDX41 binds to the 5ʹ splice site (5ʹSS) of coding RNA and coordinates RNA splicing and transcriptional elongation; loss of DDX41 prevents splicing-coupled transient pausing of RNA polymerase II at 5ʹSS, causing aberrant R-loop formation and transcription-replication collisions. Although the degree of DNA replication stress acquired in S phase is small, cells undergo mitosis with under-replicated DNA being remained, resulting in micronuclei formation and significant DNA damage, thus leading to impaired cell proliferation and genomic instability. These processes may be responsible for disease phenotypes associated with DDX41 mutations.

We report here that a defect of the interleukin common gamma subunit (gamma c) in X-linked severe combined immunodeficiency (XSCID) previously known as a missense mutation resulted instead in exon skipping in a Japanese XSCID patient. The phenotype of the patient was consistent with that of typical XSCID, and his Epstein-Barr virus-transformed B cells accordingly entirely lacked surface expression of gamma c . On analysis by the reverse transcription-polymerase chain reaction (RT-PCR), a single but small gamma c mRNA species was detected. Exon 6, which encodes the transmembrane domain of gamma c, was skipped in the mRNA. A G to A mutation was found at the last nucleotide of exon 6 of the gamma c gene (868G--A). The predicted consequence of the exon skipping is a frameshift resulting in a premature stop codon, and the mutated gamma c presumably loses association with the cell membrane. In XSCID, this mutation (868G--A) is known as a missense mutation that results in R285Q [corrected]. Previously reported patients with the same mutation apparently had no aberrant or alternative splicing but did have the R285Q [corrected] exchange. Similar mutations at the last nucleotide of an outskipped exon have been reported. However, such mutations do not always cause exon skipping. Analyses of RNA structural changes induced by the mutations supported the variability of consequences of the mutations. Taken together, our findings suggest that the 868G--A mutation of the gamma c gene may affect gamma c transcripts differently, i.e., generating missense or exon skipping, in XSCID patients with the same mutation. Patient-specific variation in splicing thus appears to occur.

New prp (pre-mRNA processing) mutants of the fission yeast Schizosaccharomyces pombe were isolated from a bank of 700 mutants that were either temperature sensitive (ts-) or cold sensitive (cs-) for growth. The bank was screened by Northern blot analysis with probes complementary to S. pombe U6 small nuclear RNA (sn RNA), the gene for which has a splicesomal (mRNA-type) intron. We identified 12 prp mutants that accumulated the U6 snRNA precursor at the nonpermissive temperature. All such mutants were also found to have defects in an early step of TFIID pre-mRNA splicing at the nonpermissive temperature. Complementation analyses showed that seven of the mutants belong to six new complementation groups designated as prp8 and prp10-prp14, whereas the five other mutants were classified into the known complementation groups prp1, prp2 and prp3. Interestingly, some of the isolated prp mutants produced elongated cells at the nonpermissive temperature, which is a phenotype typical of cell division cycle (cdc) mutants. Based on these findings, we propose that some of the wild-type products from these prp+ genes play important roles in the cellular processes of pre-mRNA splicing and cell cycle progression.

The small nuclear RNAs (snRNAs) are a class of metabolically stable small RNAs present in the nuclei of eukaryotic cells. In mammalian cells, there are six major molecular species (U1 to U6 snRNA), which are complexed with proteins, forming small nuclear ribonucleoprotein particles, snRNPs. Of these, the U1, U2, U4, U5, U6 snRNPs are thought to participate in pre-mRNA splicing as part of the spliceosome. Here, we describe the characterization of the gene coding for the Schizosaccharomyces pombe U6 snRNA. Unexpectedly, the Schiz. pombe U6 RNA gene was found to contain an intron-like sequence of 50 base pairs. Northern blot analysis and RNA sequencing revealed that this intron-like sequence is precisely removed from the transcript. The mature U6 RNA of Schiz. pombe has 77% sequence homology with the mammalian U6 RNA. In Schiz. pombe, it is possible that U6 RNA is not only involved in pre-mRNA splicing, but is also a splicing substrate. This is the first report of an intron in a snRNA gene.