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.
Department of Biochemistry and Biophysics, Program in Molecular Biology and Biotechnology, CB 3280, University of North Carolina, Chapel Hill, NC 27599, USA.
Xiao-cui
XC
Yang
Bing
B
Xu
Ivan
I
Sabath
Lalitha
L
Kunduru
Brandon D
BD
Burch
William F
WF
Marzluff
Zbigniew
Z
Dominski
D000595:Amino Acid Sequence; D051017:Apoptosis Regulatory Proteins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004720:Endonucleases / Q000378:metabolism*; D005092:Exonucleases / Q000378:metabolism*; D006657:Histones / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D011485:Protein Binding; D025941:Protein Interaction Mapping; D012322:RNA Precursors / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D016601:RNA-Binding Proteins / Q000378:metabolism; D020885:Ribonucleoprotein, U7 Small Nuclear / Q000378:metabolism
D051017:Apoptosis Regulatory Proteins; C485604:CASP8AP2 protein, human; D002135:Calcium-Binding Proteins; D006657:Histones; D012322:RNA Precursors; D016601:RNA-Binding Proteins; D020885:Ribonucleoprotein, U7 Small Nuclear; D004720:Endonucleases; D005092:Exonucleases
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