Spatial regulation of the KH domain RNA-binding protein Rnc1 mediated by a Crm1-independent nuclear export system in Schizosaccharomyces pombe.

RNA-binding proteins (RBPs) play important roles in the posttranscriptional regulation of gene expression, including mRNA stability, transport and translation. Fission yeast rnc1+ encodes a K Homology (KH)-type RBP, which binds and stabilizes the Pmp1 MAPK phosphatase mRNA thereby suppressing the Cl- hypersensitivity of calcineurin deletion and MAPK signaling mutants. Here, we analyzed the spatial regulation of Rnc1 and discovered a putative nuclear export signal (NES)Rnc1 , which dictates the cytoplasmic localization of Rnc1 in a Crm1-independent manner. Notably, mutations in the NESRnc1 altered nucleocytoplasmic distribution of Rnc1 and abolished its function to suppress calcineurin deletion, although the Rnc1 NES mutant maintains the ability to bind Pmp1 mRNA. Intriguingly, the Rnc1 NES mutant destabilized Pmp1 mRNA, suggesting the functional importance of the Rnc1 cytoplasmic localization. Mutation in Rae1, but not Mex67 deletion or overproduction, induced Rnc1 accumulation in the nucleus, suggesting that Rnc1 is exported from the nucleus to the cytoplasm via the mRNA export pathway involving Rae1. Importantly, mutations in the Rnc1 KH-domains abolished the mRNA-binding ability and induced nuclear localization, suggesting that Rnc1 may be exported from the nucleus together with its target mRNAs. Collectively, the functional Rae1-dependent mRNA export system may influence the cytoplasmic localization and function of Rnc1.

Role of the RNA-binding protein Nrd1 in stress granule formation and its implication in the stress response in fission yeast.

We have previously identified the RNA recognition motif (RRM)-type RNA-binding protein Nrd1 as an important regulator of the posttranscriptional expression of myosin in fission yeast. Pmk1 MAPK-dependent phosphorylation negatively regulates the RNA-binding activity of Nrd1. Here, we report the role of Nrd1 in stress-induced RNA granules. Nrd1 can localize to poly(A)-binding protein (Pabp)-positive RNA granules in response to various stress stimuli, including heat shock, arsenite treatment, and oxidative stress. Interestingly, compared with the unphosphorylatable Nrd1, Nrd1(DD) (phosphorylation-mimic version of Nrd1) translocates more quickly from the cytoplasm to the stress granules in response to various stimuli; this suggests that the phosphorylation of Nrd1 by MAPK enhances its localization to stress-induced cytoplasmic granules. Nrd1 binds to Cpc2 (fission yeast RACK) in a phosphorylation-dependent manner and deletion of Cpc2 affects the formation of Nrd1-positive granules upon arsenite treatment. Moreover, the depletion of Nrd1 leads to a delay in Pabp-positive RNA granule formation, and overexpression of Nrd1 results in an increased size and number of Pabp-positive granules. Interestingly, Nrd1 deletion induced resistance to sustained stresses and enhanced sensitivity to transient stresses. In conclusion, our results indicate that Nrd1 plays a role in stress-induced granule formation, which affects stress resistance in fission yeast.

Scintigraphic detection of 125I seeds after permanent brachytherapy for prostate cancer.

UNLABELLED: The purpose of this investigation was to monitor the localization and migration of 125I seeds after permanent brachytherapy for prostate cancer using a new scintigraphic technique that may overcome the drawbacks of conventional x-ray methods. METHODS: 125I seeds emit gamma-rays with an average energy peak of 28 keV. We used a gamma-camera equipped with low-energy high-resolution collimators that were tuned to an energy level of 35 keV with a 70% window width. Sixteen patients with prostate cancer were examined after 125I seed insertion. The number of seeds remaining in the prostate was confirmed using pelvic CT for postoperative dose planning; however, seeds that had migrated outside the prostate could not be detected. Furthermore, the migrated seeds were not completely traceable using chest or abdominal radiography. Thus, we adopted a scintigraphic technique to perform this task. The evaluation of radiography and scintigraphy findings was masked, and the rates of migrated seed detection were statistically examined using the McNemar test. To localize the migrated seeds, we fused the scintigraphic images of the migrated seeds and the patients' contours. RESULTS: Scintigraphy was successfully used to detect 20 migrated seeds of a total of 1,182 implanted seeds, whereas radiography was successfully used to detect 7. The sensitivity of the scintigraphy results was 20 of 20 (100%), whereas that of the radiography results was 7 of 20 (35%). Seed migration was detected in 11 of 16 patients (69%) using scintigraphy, whereas seed migration was detected in only 4 patients (25%) using radiography; this difference was statistically significant (P = 0.016). CONCLUSION: Scintigraphy is more effective for detecting seed migration and monitoring the localization of 125I seeds than radiography. The precise anatomic location of migrated seeds can be pinpointed using fusion images. Scintigraphy may become a standard procedure for monitoring seed migration during 125I brachytherapy in patients with prostate cancer.