A conserved interaction that is essential for the biogenesis of histone locus bodies.

Nuclear protein, ataxia-telangiectasia locus (NPAT) and FLICE-associated huge protein (FLASH) are two major components of discrete nuclear structures called histone locus bodies (HLBs). NPAT is a key co-activator of histone gene transcription, whereas FLASH through its N-terminal region functions in 3' end processing of histone primary transcripts. The C-terminal region of FLASH contains a highly conserved domain that is also present at the end of Yin Yang 1-associated protein-related protein (YARP) and its Drosophila homologue, Mute, previously shown to localize to HLBs in Drosophila cells. Here, we show that the C-terminal domain of human FLASH and YARP interacts with the C-terminal region of NPAT and that this interaction is essential and sufficient to drive FLASH and YARP to HLBs in HeLa cells. Strikingly, only the last 16 amino acids of NPAT are sufficient for the interaction. We also show that the C-terminal domain of Mute interacts with a short region at the end of the Drosophila NPAT orthologue, multi sex combs (Mxc). Altogether, our data indicate that the conserved C-terminal domain shared by FLASH, YARP, and Mute recognizes the C-terminal sequence of NPAT orthologues, thus acting as a signal targeting proteins to HLBs. Finally, we demonstrate that the C-terminal domain of human FLASH can be directly joined with its N-terminal region through alternative splicing. The resulting 190-amino acid MiniFLASH, despite lacking 90% of full-length FLASH, contains all regions necessary for 3' end processing of histone pre-mRNA in vitro and accumulates in HLBs.

Factors that affect consultation and screening for fecal incontinence.

BACKGROUND & AIMS: Fecal incontinence (FI) affects 15% of people age 70 years and older, but only 10% to 30% discuss FI with their physicians. We aimed to identify barriers that prevent people from consulting with their physicians, and that prevent physicians from screening for FI. METHODS: We performed structured interviews of 124 individuals with FI (mean age, 56 y; 87.9% women) recruited from 6 medical offices at the University of North Carolina Hospitals from June 2012 through March 2013. The subjects completed the Fecal Incontinence Severity Index and the Fecal Incontinence Quality of Life Scale questionnaires. Interview questions aimed to determine which patients had consulted physicians for FI. Eleven of the 56 physicians with patients included in the study responded to the survey. RESULTS: Eighty-eight of the 124 participants consulted with their physicians about FI (consulters). These individuals had a higher incidence of depression than the 36 subjects who did not consult with their physicians about FI (nonconsulters; P = .04), but similar Fecal Incontinence Severity Index scores. A smaller proportion of nonconsulters were aware of available treatments than consulters (P < .01). Fifty-six percent of nonconsulters said their FI was not serious enough to consult a physician. There was no difference between consulters and nonconsulters in embarrassment in talking about FI. Among consulters, 88% initiated the conversation about FI with their physician. Seven of the 11 responding physicians screened for FI, but only screened high-risk patients. The 4 physicians who did not screen for FI were unaware of its prevalence, viewed FI as a low priority, or stated that patients were responsible for reporting their own symptoms. CONCLUSIONS: Based on surveys of physicians and patients, many patients have insufficient knowledge about the availability and effectiveness of treatments for FI. Some people with FI do not discuss it with their physician because their symptoms are mild, and most prefer physicians to ask them directly about FI. Educating patients and physicians about the prevalence of FI and management strategies may improve consultation rates.

FLASH is required for the endonucleolytic cleavage of histone pre-mRNAs but is dispensable for the 5' exonucleolytic degradation of the downstream cleavage product.

3'-end cleavage of histone pre-mRNAs is catalyzed by CPSF-73 and requires the interaction of two U7 snRNP-associated proteins, FLASH and Lsm11. Here, by using scanning mutagenesis we identify critical residues in human FLASH and Lsm11 that are involved in the interaction between these two proteins. We also demonstrate that mutations in the region of FLASH located between amino acids 50 and 99 do not affect binding of Lsm11. Interestingly, these mutations convert FLASH into an inhibitory protein that reduces in vitro processing efficiency of highly active nuclear extracts. Our results suggest that this region in FLASH in conjunction with Lsm11 is involved in recruiting a yet-unknown processing factor(s) to histone pre-mRNA. Following endonucleolytic cleavage of histone pre-mRNA, the downstream cleavage product (DCP) is degraded by the 5'-3' exonuclease activity of CPSF-73, which also depends on Lsm11. Strikingly, while cleavage of histone pre-mRNA is stimulated by FLASH and inhibited by both dominant negative mutants of FLASH and anti-FLASH antibodies, the 5'-3' degradation of the DCP is not affected. Thus, the recruitment of FLASH to the processing complex plays a critical role in activating the endonuclease mode of CPSF-73 but is dispensable for its 5'-3' exonuclease activity. These results suggest that CPSF-73, the catalytic component in both reactions, can be recruited to histone pre-mRNA largely in a manner independent of FLASH, possibly by a separate domain in Lsm11.