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
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
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.
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)
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.
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-)
© copyright 2006-2023, Liang Tong.