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.
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-specific 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.
Yang
Xiao-cui
XC
Program in Molecular Biology and Biotechnology, University of North Carolina, Chapel Hill, NC 27599, USA.
Torres
Matthew P
MP
Marzluff
William F
WF
Dominski
Zbigniew
Z
D020413:3' Untranslated Regions / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D045744:Cell Line, Tumor; D006657:Histones / Q000235:genetics*; D047468:Immunoprecipitation; D051379:Mice; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D020038:RNA, Spliced Leader / Q000235:genetics / Q000378:metabolism; D020885:Ribonucleoprotein, U7 Small Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D020413:3' Untranslated Regions; D006657:Histones; D012322:RNA Precursors; D020038:RNA, Spliced Leader; D020885:Ribonucleoprotein, U7 Small Nuclear