- R. A. Koupet al.,Nat. Med. 27 , 1493–1494 (2021).
- J. Kerteset al.,medRxiv2021.09.01.21262957 [Preprint]
(2021). https://doi.org/10.1101/2021.09.01.21262957. - M. P. Andrasiket al.,PLOS ONE 16 , e0258858 (2021).
- P. B. Gilbertet al., COVID-19 Prevention Network Immune
Correlates Analyses, version 1.1.1, Zenodo (2021);
https://doi.org/10.5281/zenodo.5593130.
ACKNOWLEDGMENTS
We thank the volunteers who participated in the COVE trial.
We also thank J. Mascola, M. Hepburn, R. A. Johnson, M. Marovich,
and M. Robb.Funding:This study was supported by the Public
Health Service; the National Institute of Allergy and Infectious
Diseases; National Institutes of Health grant UM1AI068635 (to
P.B.G.); the Office of Research Infrastructure Programs; National
Institutes of Health grant (S10OD028685) (Fred Hutchinson
Cancer Research Center); Moderna, Inc. Federal funds from the
Office of the Assistant Secretary for Preparedness and Response;
the Biomedical Advanced Research and Development Authority,
contract no. 75A50120C00034 (Moderna, Inc.); and the Infectious
Disease Clinical Research Consortium Leadership Group through
the National Institute for Allergy and Infectious Diseases,
grant UM1 AI148684-03 (to K.M.N.).Author contributions:
Conceptualization: P.B.G., D.C.M., A.B.M., Y.F., D.B., D.F., R.O.D.,
and R.A.K. Data curation: P.B.G., Y.F., D.B., W.D., H.Z., Y.L., C.Y.,
B.B., L.W.P.v.d.L., and N.S.H. Formal analysis: P.B.G., Y.F., D.B.,
Y.L., C.Y., B.B., L.W.P.v.d.L., and N.S.H. Funding acquisition: P.B.G.,
R.O.D., and R.A.K. Investigation: D.C.M., A.B.M., K.M., L.J., F.C.,
B.F., B.C.L., S.O., C.M., A.E., and M.S.-K. Methodology: P.B.G., Y.F.,
D.B., L.W.P.v.d.L., and D.F. Project administration: C.R.H., S.O.,
M.S.-K., C.H., R.O.D., and R.A.K. Resources: D.C.M., A.B.M., J.M.,
H.M.E.S., L.R.B., M.B., L.D.L.C., C.G., S.K., C.F.K., M.P.A., J.G.K.,
L.C., K.M.N., R.P., R.O.D., and R.A.K. Software: P.B.G., Y.F., D.B.,
Y.L., C.Y., B.B., L.W.P.v.d.L., and N.S.H. Supervision: P.B.G., D.C.M.,
A.B.M., Y.F., D.B., D.F., R.O.D., and R.A.K. Validation: P.B.G., Y.F.,
and D.B. Visualization: P.B.G., Y.F., D.B., Y.L., B.B., L.W.P.v.d.L.,
and L.N.C. Writing–original draft: P.B.G. and L.N.C. Writing–
review and editing: All coauthors.Competing interests:All
authors have completed the ICMJE uniform disclosure form at
http://www.icmje.org/downloads/coi_disclosure.docx and declare:
P.B.G., D.C.M., Y.F., C.M., A.E., M.S.-K., Y.L., C.Y., B.B., L.W.P.v.d.L.,
H.M.E.S., L.R.B., M.B., C.F.K., M.P.A., L.C., and L.N.C. had support
(in the form of grant payments to their institution) from the
National Institutes of Health for the submitted work; R.A.K. had
support (in the form of funds to the VRC to cover assay
performance) from the National Institutes of Health for the
submitted work; M.B., L.D.L.C., and C.F.K. had support (in the form
of payments to their institution) from Moderna for the submitted
work; W.D., H.Z., J.M., and R.P. are employed by Moderna Tx, Inc.
and have stock and/or stock options in Moderna Tx, Inc.; L.R.B.
declares support (in the form of grant payments to his institution)
within the previous 3 years from the National Institutes of Health,
the Wellcome Trust, and the Bill and Melinda Gates Foundation and
has served on NIAID-NIH SMCs within the previous 3 years; M.B.
declares support (in the form of grant payments to her institution)
within the previous 3 years from the National Institutes of Health
and the Centers for Disease Control; C.F.K. declares support (in
the form of grant payments to her institution) within the previous
3 years from the National Institutes of Health, the Centers for
Disease Control, Humanigen, Novavax, Viiv, and Gilead, as well as
payments from Medscape and from Clinical Care Options within
the previous 3 years for educational events; K.M.N. declares
support (in the form of grant payments to her institution) within
the previous 3 years from GSK, Pfizer, the Bill and Melinda Gates
Foundation, and PATH for vaccine research, as well as support
(in the form of grant payments to her institution) within the
previous 3 years from the National Institutes of Health for influenza
vaccine research; and D.C.M. declares support from Moderna
within the previous 3 years for work outside the scope of the
submitted work. All other authors declare no support from any
organization for the submitted work, no financial relationships with
any organizations that might have an interest in the submitted
work in the previous 3 years, and no other relationships or
activities that could appear to have influenced the submitted work.
Data and materials availability:As the trial is ongoing, access to
participant-level data and supporting clinical documents with
qualified external researchers may be available upon request and is
subject to review once the trial is complete. The code is publicly
available at Zenodo ( 38 ). This work is licensed under a Creative
Commons Attribution 4.0 International (CC BY 4.0) license, which
permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited. To view a copy
of this license, visit https://creativecommons.org/licenses/by/4.0/.
This license does not apply to figures/photos/artwork or
other content included in the article that is credited to a
third party; obtain authorization from the rights holder before
using such material.SUPPLEMENTARY MATERIALS
science.org/doi/10.1126/science.abm3425
Materials and Methods
Supplementary Text
Figs. S1 to S30
Tables S1 to S9
Immune Assays Team Collaborator ListModerna, Inc. Team Collaborator List
CoVPN/COVE Team Collaborator List
USG/CoVPN Biostatistics Team Collaborator List
References ( 39 – 53 )
MDAR Reproducibility Checklist
Statistical Analysis Plan16 September 2021; accepted 16 November 2021
Published online 23 November 2021
10.1126/science.abm3425SPLICEOSOMEStructural basis of branch site recognition
by the human spliceosome
Jonas Tholen1,2 , Michal Razew^1 , Felix Weis^3 , Wojciech P. Galej^1 *Recognition of the intron branch site (BS) by the U2 small nuclear ribonucleoprotein (snRNP) is a critical
event during spliceosome assembly. In mammals, BS sequences are poorly conserved, and unambiguous
intron recognition cannot be achieved solely through a base-pairing mechanism. We isolated human
17 SU2 snRNP and reconstituted in vitro its adenosine 5 ́-triphosphate (ATP)–dependent remodeling and
binding to the pre–messenger RNA substrate. We determined a series of high-resolution (2.0 to
2.2 angstrom) structures providing snapshots of the BS selection process. The substrate-bound U2
snRNP shows that SF3B6 stabilizes the BS:U2 snRNA duplex, which could aid binding of introns with
poor sequence complementarity. ATP-dependent remodeling uncoupled from substrate binding captures
U2 snRNA in a conformation that competes with BS recognition, providing a selection mechanism
based on branch helix stability.R
emoval of introns from pre-mRNA is
catalyzed by a large and dynamic RNA-
protein complex known as the spliceo-
some. The spliceosome is assembled de
novo on each pre-mRNA substrate from
five small nuclear ribonucleoprotein (snRNP)
particles and several dozen protein factors.
During spliceosome assembly, three conserved
positions in the pre-mRNA—the 5′splice site
(5′-SS), branch site (BS), and 3′splice site (3′-SS)—
are specifically recognized by the components
of the spliceosome, allowing a two-step trans-
esterification reaction to occur.
In mammalian cells, the BS is initially rec-
ognized by SF1 (mBBP) in cooperation with
U2AF2 (U2AF65), which binds the polypyrim-
idine tract (PPT) sequence ( 1 ). Concomitantly,
U1 snRNP binds to the 5′-SS and together they
form the first, adenosine 5 ́-triphosphate (ATP)–
independent spliceosome assembly interme-
diate known as complex E ( 2 ). The U2 snRNP
is loosely associated with complex E ( 3 ), and
its stable incorporation into the prespliceosome
(complex A) requires ATP and formation of
base-pairing interactions between the BS and
the U2 snRNA ( 4 ).In yeast, several factors have been shown to
facilitate complex A formation. Among them
are Cus2 (human HTATSF1) ( 5 )andtheRNA-
dependent DEAD-box ATPase, Prp5 (human
DDX46), whose activity is required for Cus2
displacement ( 6 – 8 ) and fidelity control of BS
recognition ( 9 , 10 ).
During BS recognition, an evolutionarily
conserved branchpoint-interacting stem loop
(BSL) presents U2 nucleotides to the intron
BS for base pairing ( 11 , 12 ). Although branch
helix formation is subject to a fidelity check-
point, it is unclear how branch helix stability is
sensed by the splicing machinery ( 9 , 10 ).
Upon engagement of U2 snRNA with the
pre-mRNA substrate, a 15–nucleotide (nt)–
long branch helix is formed, adopting helical
geometry even in the absence of full comple-
mentarity. The length of the branch helix is con-
served between yeast and human spliceosomes
and maintained throughout different stages of
splicing ( 13 – 15 ). In early splicing complexes,
the branch helix is accommodated within a cav-
ity formed by the heteroheptameric SF3b com-
plex ( 7 ), which contacts the pre-mRNA around
the BS and stabilizes the U2 snRNA:BS base-
pairing interaction ( 16 , 17 ). The branchpoint
adenosine (BP-A) is bulged out of the branch
helix and binds into a pocket formed by SF3B1
and PHF5A ( 13 , 14 ). Mutations in SF3B1 asso-
ciated with myelodysplastic syndromes have
been shown to modulate BS selection ( 18 , 19 ).50 7 JANUARY 2022•VOL 375 ISSUE 6576 science.orgSCIENCE
(^1) European Molecular Biology Laboratory, 71 Avenue des
Martyrs, 38042 Grenoble, France.^2 Heidelberg University,
Faculty of Biosciences, Heidelberg, Germany.^3 European
Molecular Biology Laboratory, Structural and Computational
Biology Unit, Meyerhofstraße 1, 69117 Heidelberg, Germany.
*Corresponding author. Email: [email protected]
†Candidate for Joint PhD degree from EMBL and Heidelberg University.
RESEARCH | RESEARCH ARTICLES