SF3B14 is involved in the centrosome regulation through splicing of TUBGCP6 pre-mRNA.

Splicing precursor messenger RNA (pre-mRNA) is a critical step to produce physiologically functional protein. Splicing failure not only gives rise to dysfunctional proteins but also generates abnormal protein function, which causes several diseases. Several pre-mRNA splicing factors are reported to regulate mitosis directly at mitotic structures and/or indirectly through controlling the pre-mRNA splicing for mitotic proteins. In this study, we described the mitotic functions of SF3B14, a component of the spliceosomal U2 small nuclear ribonucleoprotein (snRNP), which we identified as a candidate involved in mitosis based on the large-scale RNA interference (RNAi) screen of the nucleolar proteome database. We observed that SF3B14 depletion caused prolonged mitosis and several mitotic defects, such as monopolar spindle and chromosome misalignment during metaphase. Although SF3B14 was found in the nucleolar proteome database, our immunofluorescent stainings demonstrated that SF3B14 was predominantly localized in the nucleoplasm and excluded from the nucleolus during interphase. In addition, SF3B14 did not colocalize with specific mitotic structures during mitosis, which is not in line with its direct mitotic function. Notably, we found that the SF3B14 depletion reduced protein levels of TUBGCP6, required for centrosome regulation, and increased the unspliced/spliced ratio of its mRNA. Taken together, we propose that the pre-mRNA of TUBGCP6 is one of the targets for SF3B14 splicing through which SF3B14 controls mitotic chromosome behavior.

Detection of 30 bp DNA fragments with a sensitive modified Southern blot analysis.

To evaluate crops generated by new breeding techniques, it is important to confirm the removal of recombinant DNAs (rDNAs) derived from foreign genes including unintentionally introduced short rDNA(s). We attempted to develop a sensitive detection method for such short rDNAs using Southern blot analysis and performed a model study targeting single-copy endogenous genes in plants. To increase the detection sensitivity, the general protocol for Southern blot analysis was modified. In the model study, we used endogenous-gene-targeting probes in which complementary sequences were serially replaced by dummy sequences, and detected complementary sequences as well as 30 bp. We further evaluated the sensitivity using short rDNAs derived from GM sequences as pseudoinsertions, and the results demonstrated that rDNA-insertions as small as 30 bp could be detected. The results suggested that unintentionally introduced rDNA-insertions were 30 bp or more in length could be detected by the Southern blot analysis.