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Molecular and Cellular Biology, August 2009, p. 4045-4056, Vol. 29, No. 15
0270-7306/09/$08.00+0     doi:10.1128/MCB.00296-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Three Proteins of the U7-Specific Sm Ring Function as the Molecular Ruler To Determine the Site of 3'-End Processing in Mammalian Histone Pre-mRNA {triangledown} ,{dagger}

Xiao-cui Yang, Matthew P. Torres, William F. Marzluff, and Zbigniew Dominski*

Department of Biochemistry and Biophysics and Program in Molecular Biology and Biotechnology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599

Received 6 March 2009/ Returned for modification 1 April 2009/ Accepted 19 May 2009

Cleavage of histone pre-mRNAs at the 3' end is guided by the U7 snRNP, which is a component of a larger 3'-end processing complex. To identify other components of this complex, we isolated proteins that stably associate with a fragment of histone pre-mRNA containing all necessary processing elements and a biotin affinity tag at the 5' end. Among the isolated proteins, we identified three well-characterized processing factors: the stem-loop binding protein (SLBP), which interacts with the stem-loop structure upstream of the cleavage site, and both Lsm11 and SmB, which are components of the U7-specifc Sm ring. We also identified 3'hExo/Eri-1, a multifunctional 3' exonuclease that is known to trim the 3' end of 5.8S rRNA. 3'hExo primarily binds to the downstream portion of the stem-loop structure in mature histone mRNA, with the upstream portion being occupied by SLBP. The two proteins bind their respective RNA sites in a cooperative manner, and 3'hExo can recruit SLBP to a mutant stem-loop that itself does not interact with SLBP. UV-cross-linking studies used to characterize interactions within the processing complex demonstrated that 3'hExo also interacts in a U7-dependent manner with unprocessed histone pre-mRNA. However, this interaction is not required for the cleavage reaction. The region between the cleavage site and the U7-binding site interacts with three low-molecular-weight proteins, which were identified as components of the U7-specific Sm core: SmB, SmD3, and Lsm10. These proteins likely rigidify the substrate and function as the molecular ruler in determining the site of cleavage.

* Corresponding author. Mailing address: Program in Molecular Biology and Biotechnology, CB #3280, University of North Carolina, Chapel Hill, NC 27599. Phone: (919) 843-0307. Fax: (919) 962-1274. E-mail: dominski{at}med.unc.edu

{triangledown} Published ahead of print on 26 May 2009.

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/.

Molecular and Cellular Biology, August 2009, p. 4045-4056, Vol. 29, No. 15
0270-7306/09/$08.00+0     doi:10.1128/MCB.00296-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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