The Histone pre-mRNA 3'-end Processing Project

Updated Jan. 2023

Metazoan replication-dependent histone pre-mRNAs are cleaved but not polyadenylated at their 3' end. The cleavage site is located between the conserved stem-loop structure and a purine-rich histone downstream element (HDE). The stem-loop is recognized by stem-loop binding protein (SLBP), and 3'-5' exoribonuclease 3'hExo after the cleavage. The HDE base pairs with the 5' region of the U7 snRNA, and the U7 snRNP is critical for histone pre-mRNA processing.

The Sm ring of U7 snRNP contains two unique subunits, Lsm10 and Lsm11, in place of SmD1 and SmD2 of the spliceosomal snRNPs. Lsm11 has a 150 residue long N-terminal extension, and is crucial for interacting FLASH, which in turn recruits the histone pre-mRNA cleavage complex (HCC). Lsm10 has small extensions at its N- and C-termini, but it is not known whether they have any functions.

The HCC contains CPSF73, CPSF100, symplekin and CstF64, with CPSF73 being the endonuclease that catalyzes the cleavage reaction. The HCC is equivalent to the mammalian cleavage factor (mCF) in the canonical processing machinery, with CPSF73 as the endonuclease in both machineries.

Major findings from this project

The structure of the ternary complex of a 26-nt stem-loop RNA in complex with the RNA binding domain (RBD) of human SLBP and human 3'hExo has been determined.

Only one base of the RNA is recognized specifically by the two proteins, the guanine in the second basepair of the stem, by Arg181 of the RBD.

Three of the four bases in the loop are flipped out to interact with the proteins.

SLBP and 3'hExo primarily recognizes the shape of the stem-loop RNA.

The two proteins have no direct contact with each other in the complex. The cooperativity in their binding is mediated by induced changes in the structure of the RNA. Binding of one protein induces a conformation of the stem-loop that promotes the binding of the other protein.

The 3'-end of the stem-loop RNA is located in the active site of 3'hExo, providing insight into how this enzyme trims and degrades the histone mRNAs.

Phosphorylation of Thr171 in SLBP enhances the affinity for the stem-loop RNA, but the residue is not in the interface with the RNA. Phosphorylation may stabilize a conformation of the SLBP that is more competent for binding the RNA.

FLASH NTD is a coiled-coil dimer.

FLASH NTD-Lsm11 NTD complex has 2:1 stoichiometry.

L118A/I119A mutation in the FLASH NTD dimer interface can interfere with Lsm11 binding without disrupting dimerization.

An active human histone pre-mRNA 3'-end processing machinery has been reconstituted, using 13 proteins and 2 RNAs (U7 ring, HCC, SLBP, FLASH, U7 snRNA, H2a* histone pre-mRNA).

The structure of the active machinery has been determined by cryo-EM. The overall structure has the shape of an amphora.

The HDE-U7 duplex is surrounded by symplekin NTD, CPSF100 and CPSF73.

Symplekin NTD is required for the processing activity. Mutations in the active site of CPSF73 abolishes activity. Ssu72 inhibits processing in vitro as it competes with the binding of the HDE-U7 duplex.

CPSF73 is in an active conformation, with a 17 degree rotation of the b-CASP domain.

There is a deep channel between the metallo-b-lactamase and b-CASP domains of CPSF73 in the active conformation, which is only large enough to accommodate a single-stranded RNA.

The pre-mRNA is captured in the active site of CPSF73. The binding mode reveals the catalytic mechanism for the endonuclease activity.

The active state is triggered by the recognition of the HDE-U7 duplex, ultimately mediated by the N- and C-terminal extensions of Lsm10 contacting the b-CASP domain of CPSF73.

There is a large conformational change of HCC in this active state compared to that of mCF in the inactive state. There are close contacts between CPSF73 and CPSF100 in the active state. In fact they form a pseudo dimer.

The structure and biochemical studies provide significant insights into the histone pre-mRNA 3'-end processing cycle. The N-terminal extension of Lsm11 recruits FLASH, which in turn recruits HCC to the machinery. The recognition of the pre-mRNA leads to the activation of HCC and CPSF73, resulting in the cleavage of the pre-mRNA. CPSF73 then funtions as an exonuclease to degrade the downstream cleavage product, and the machinery can be recycled for the next pre-mRNA molecule.

Biochemical studies show that CPSF73 in the histone 3'-end processing machinery also possesses 5'-3' exonuclease activity, which can degrade the downstream cleavage product. CPSF73 also has activity toward ssDNA.

Publications from this project

D. Tan, W.F. Marzluff, Z. Dominski & L. Tong. (2013). Structure of histone mRNA stem-loop, human stem-loop binding protein and 3'hExo ternary complex. Science, 339, 318-321. Reprint(PDF)
J. Zhang, D. Tan, E.F. DeRose, L. Perera, Z. Dominski, W.F. Marzluff,* L. Tong* & T.M. Tanaka Hall.* (2014). Molecular mechanisms for the regulation of histone mRNA stem-loop-binding protein by phosphorylation. Proc. Natl. Acad. Sci. USA, 111, E2937-E2946. (*-co-corresponding authors) Reprint(PDF)
W.S. Aik, M.-H. Lin, D. Tan, A. Tripathy, W.F. Marzluff, Z. Dominski, C.-Y. Chou & L. Tong. (2017). The N-terminal domains of FLASH and Lsm11 form a 2:1 heterotrimer for histone pre-mRNA 3-end processing. PLoS One, 12, e0186034. Reprint(PDF)
Y. Sun,* Y. Zhang,* W.S. Aik, X.-C. Yang, W.F. Marzluff, T. Walz$, Z. Dominski$ & L. Tong.$ (2020). Structure of an active human histone pre-mRNA 3'-end processing machinery. Science, 367, 700-703. (*-equal first authors, $-co-corresponding authors)
K. Bucholc,* W.S. Aik,* X.-C. Yang, K. Wang, Z.H. Zhou, M. Dadlez, W.F. Marzluff, L. Tong$ & Z. Dominski.$ (2020). Composition and processing activity of a semi-recombinant holo U7 snRNP. Nucl. Acids Res. 48, 1508-1530. (Epub 12/10/19) (*-equal first authors, $-co-corresponding authors)
X.-C. Yang,* Y. Sun,* W.S. Aik, W.F. Marzluff, L. Tong$ & Z. Dominski.$ (2020). Studies with recombinant U7 snRNP demonstrate that CPSF73 is both an endonuclease and a 5'-3' exonuclease. RNA, 26, 1345-1359. (*-equal first authors, $-co-corresponding authors)
P.A. Gutierrez, K. Baughman, Y. Sun & L. Tong. (2021). A real-time fluorescence assay for CPSF73, the nuclease for pre-mRNA 3'-end processing. RNA, 27, 1148-1154.
Y. Sun, W.S. Aik, X.-C. Yang, W.F. Marzluff, Z. Dominski$ & L. Tong.$ (2021). Reconstitution and biochemical assays of an active human histone pre-mRNA 3'-end processing machinery. Methods Enzymol. 655, 292-324. ($-co-corresponding authors)
Z. Dominski$ & L. Tong$ (2021). U7 deciphered: the mechanism that forms the unusual 3' end of metazoan replication-dependent histone mRNAs. Biochem. Soc. Trans. 49, 2229-2240. ($-co-corresponding authors)

Funding for this project

NIH R01GM077175 (2007-2016)
NIH R35GM118093 (2016-)