The pre-mRNA 3'-end Processing Project
Updated Jan. 2018
Most eukaryotic messenger RNA precursors (pre-mRNAs) must undergo extensive maturational
processing, including 5'-end capping, splicing, and 3'-end cleavage and polyadenylation. The
addition of a poly(A) tail is important for mRNA stability, and enhances mRNA transport to
the cytoplasm and mRNA translation.
The 3'-end processing events include cleavage at a specific site in the pre-mRNA followed by
the addition of the poly(A) tail. In mammals, the cleavage site is defined by an upstream
and a downstream U or G/U-rich element.
A large number of protein factors have been identified that are crucial for pre-mRNA 3'-end
processing. These proteins form several sub-complexes, such as the cleavage and polyadenylation
specificity factor (CPSF) and the cleavage stimulation factor (CstF). CPSF contains 5 subunits,
CPSF-30, -73, -100, -160, and Fip1, and CstF contains 3 subunits, Cstf-50, -64, and -77. CPSF-160
recognizes the upstream AAUAAA motif, and CstF-64 recognizes the downstream U- or G/U-rich element.
Major findings from this project
- The crystal structures of human CPSF-73, and its weak sequence homolog yeast Ydh1p
(CPSF-100), have been determined.
- The structures of CPSF-73 and CPSF-100 contain an N-terminal metallo-b-lactamase domain
and a novel b-CASP domain. A segment of 60 residues after the b-CASP domain contributes to the
N-terminal metallo-b-lactamase domain.
- CPSF-73 binds two zinc ions, each in an octahedral coordination. A sulfate ion in the structure
is a good mimic of the phosphate group of the pre-mRNA substrate.
- The active site in CPSF-73 is located deep in the interface between the metallo-b-lactamase
domain and the b-CASP domain.
- Despite having a similar overall structure, the zinc ligands are absent in CPSF-100. This
subunit cannot bind zinc and is inactive.
- RNA cleavage assays show that CPSF-73 possesses predominantly non-specific endoribonuclease
- The structural and biochemical studies provide direct experimental evidence that CPSF-73
is the nuclease for the cleavage reaction of pre-mRNA 3'-end processing.
- The crystal structures of the HAT domain of murine CstF-77 as well as its HAT-C subdomain
have been determined.
- The HAT domain contains two subdomains, a HAT-N domain with 5 HAT repeats and a HAT-C domain
with 7 repeats.
- The HAT domain structure is a highly extended dimer, spanning about 165 A. The dimer interface
is extensive, and the residues in the interface are mostly conserved.
- Analytical ultracentrifugation and yeast two-hybrid studies confirm that HAT domain can
dimerize in solution.
- The structural, biochemical and biophysical studies suggest CstF-77 may dimerize during
its function in pre-mRNA 3'-end processing.
The structure of yeast Rna14-Rna15 complex shows a conserved dimeric assocation of the HAT
domain in Rna14.
- The structure of the human symplekin N-terminal domain in complex with Ssu72 and a
Pol II CTD phosphopeptide has been determined at 2.4A resolution.
- Symplekin N-terminal domain contains seven pairs of anti-parallel helices, with
a backbone fold similar to HEAT/Arm repeats.
- Ssu72 is bound to the concave face of symplekin.
- The CTD phosphopeptide is bound with the pSer5-Pro6 peptide bond in the cis configuration,
indicating that Ssu72 can only dephosphorylate the cis configuration of this bond, in
contrast to current hypothesis. Ssu72 is the first phosphatase known to have a specificity
for the cis configuration.
- The active site of Ssu72 is located 25A away from the interface with symplekin. However,
symplekin can stimulate the phosphatase activity of Ssu72. Therefore, symplekin may be more
than just a passive scaffold, but instead may actively regulate the catalysis by Ssu72.
- The N-terminal domain of symplekin inhibits transcription-coupled pre-mRNA 3'-end
processing. Ssu72 can block this inhibition, demonstrating for the first time a role for
mammalian Ssu72 in pre-mRNA 3'-end processing.
- An active site mutant of Ssu72 cannot block this inhibition, suggesting that
the phosphatase activity is required for 3'-end processing.
Ssu72 recognizes pSer7 CTD in the opposite orientation compared to pSer5 phosphopeptide.
Ssu72 has much weaker phosphatase activity toward pSer7 compared to pSer5, based on
phosphatate release assays.
Rtr1 is a novel zinc finger protein, but its structure lacks an active site and it does not
have pSer5 phosphatase activity.
Replication-dependent histone pre-mRNAs are cleaved but not polyadenylated. Their processing
uses a different machinery. They contain a conserved stem-loop near the 3'-end, which is
recognized by stem-loop binding protein (SLBP) and 3'-5' exoribonuclease 3'hExo.
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.
The CTDs of IntS9 and IntS11 have extensive interactions, involving highly conserved residues.
Mutations in the IntS9-IntS11 interface can
block their interactions and Integrator function.
The proper interaction of IntS9 and IntS11 is important for Integrator function.
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.
Publications from this project
Mandel CR, Kaneko S, Zhang H, Gebauer D, Vethantham V, Manley JL, Tong L. (2006).
Polyadenylation factor CPSF-73 is the pre-mRNA 3'-end-processing endonuclease.
Nature, 444, 953-956.
Bai Y, Auperin TC, Chou C-Y, Chang G-G, Manley JL, Tong L. (2007).
Crystal structure of murine CstF-77: dimeric association and implications for
polyadenylation of mRNA precursors.
Mol. Cell. 25, 863-875.
Mandel CR, Gebauer D, Zhang H, Tong L. (2006).
A serendipitous discovery that in situ proteolysis is essential for
the crystallization of yeast CPSF-100 (Ydh1p).
Acta Cryst. F62, 1041-1045.
Bai Y, Auperin TC, Tong L. (2007).
The use of in situ proteolysis in the crystallization of murine CstF-77.
Acta Cryst. F63, 135-138.
Mandel CR, Tong L. (2007).
How to get all "A"s in polyadenylation.
Structure. 15, 1024-1026.
Mandel CR, Bai Y, Tong L. (2008).
Protein factors in pre-mRNA 3'-end processing
Cell. Mol. Life Sci.. 65, 1099-1122.
K. Xiang, T. Nagaike,* S. Xiang,* T. Kilic, M.M. Beh, J.L. Manley
L. Tong. (2010). Crystal structure of the human symplekin-Ssu72-CTD
Nature. 467, 729-733.
Y. Bai, S.K. Srivastava, J.H. Chang, J.L. Manley L. Tong. (2011).
Structural basis for dimerization and activity of human PAPD1,
a noncanonical poly(A) polymerase.
Mol. Cell. 41, 311-320.
J.H. Chang & L. Tong. (2012). Mitochondrial poly(A) polymerase
and polyadenylation. Biochim. Biophys. Acta, 1819, 992-997.
A.R. Paulson & L. Tong. (2012). Crystal structure of the Rna14-Rna15
complex. RNA, 18, 1154-1162.
K. Xiang, J.L. Manley & L. Tong. (2012). The yeast regulator of
transcription protein Rtr1 lacks an active site and phosphatase
activity. Nature Commun. 3, 946. doi: 10.1038/ncomms1947.
K. Xiang, J.L. Manley & L. Tong. (2012). An unexpected binding
mode for a Pol II CTD peptide phosphorylated at Ser7 in the active
site of the CTD phosphatase Ssu72. Genes Develop. 26, 2265-2270.
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.C. Wilson, H.-T. Hornig-Do, F. Bruni, J.H. Chang, A.A. Jourdain,
J.-C. Martinou, M. Falkenberg, H. Spahr, N.-G. Larsson, R.J. Lewis,
L. Hewitt, A. Basle, H.E. Cross, L. Tong, R.R. Lebel, A.H. Crosby,
Z.M.A. Chrzanowska-Lightowlers* & R.N. Lightowlers.* (2014).
A human mitochondrial poly(A) polymerase mutation reveals the
complexities of post-transcriptional mitochondrial gene expression.
Human Mol. Genet. 23, 6345-6355. (*-co-corresponding authors)
A.R. Jurado, D. Tan, X. Jiao, M. Kiledjian & L. Tong. (2014).
Structure and function of pre-mRNA 5'-end capping quality control
and 3'-end processing.
Biochem. 53, 1882-1898.
K. Xiang, L. Tong & J.L. Manley. (2014).
Delineating the structural blueprint of the pre-mRNA 3' end processing
Mol. Cell. Biol. 34, 1894-1910.
Y. Wu,* T.R. Albrecht,* D. Baillat, E.J. Wagner$ & L. Tong.$ (2017).
Molecular basis for the interaction between Integrator subunits
IntS9 and IntS11 and its functional importance.
Proc. Natl. Acad. Sci. USA, 114, 4394-4399.
(*-equal first authors, $-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.
Funding for this project
NIH R01GM077175 (2007-2016)
NIH R35GM118093 (2016-)
© copyright 2006-2018, Liang Tong.