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Significance of the First Transcribed Nucleoside
upon Interaction with the Human Nuclear
Cap-Binding Complex Remigiusz Worch1, Anna
Niedzwiecka1,3, Janusz Stepinski 1, Marzena
Jankowska-Anyszka2, Catherine Mazza4, Edward
Darzynkiewicz 1, Stephen Cusack4 and Ryszard
Stolarski1 1Department of Biophysics and 2Faculty
of Chemistry, Warsaw University, Poland,
3Biological Physics Group, Institute of Physics
PAS, Warsaw, 4EMBL, Grenoble, France
Introduction All nuclear transcribed mRNAs
possess a 5'-terminal "cap", m7GpppN, in which
7-methylguanosine is linked by a
5-5-triphosphate bridge to the first
transcribed nucleoside. Recognition of the mRNA
5 cap structure by the nuclear protein Cap
Binding Complex (CBC) plays a key role in
pre-mRNA splicing, polyadenylation of the 3'
terminus, U snRNA transport, nonsense-mediated
decay and translation initiation. The CBC is a
heterodimer consisting of two subunits CBP20 and
CBP80. The former directly interacts with the cap
structure while the latter is essential for the
active complex formation. The previously known
crystal structures of the apo-CBC (1) and the CBC
in the complex with a cap analogue, m7GpppG
(2,3), showed large conformational changes of the
N- and C- terminal parts of CBP20 upon mRNA 5cap
binding. The m7G moiety of 5 mRNA cap interacts
with the CBC by sanwich stacking with Tyr20 and
Tyr 43 enhanced by numerous hydrogen bonds, and
the first transcribed nucleoside stacks with Tyr
138 (Scheme 1).  While the 3D structure of the
protein was solved, the consistent energetic
description of the cap-CBC interaction was
lacking. To address the mechanistic bases of
complex formation and stability, solution studies
were necessary. Because the CBC has many
fluorescent residues and cap binding results in
quenching of the intrinsic protein fluorescence
due to conformational changes, a method of
time-synchronized titration (4) was used in order
to analyze the molecular basis of the cap
affinity for the CBC. By means of this method,
including the activity of the non-enzymatic
protein and emission of all species explicitly
into the analysis, we have determined equilibrium
association constants (Kas) for a series of mono-
and dinucleotide cap analogues (Scheme 2) and
with a tetranucleotide mRNA fragment,
m7GpppAm2pUm2pAm2. The Kas values can be
interpreted in terms of Gibbs free energy of
binding (?G? ).  The role of stacking between
Tyr138 of the CBC and the base moiety of the
first transcribed nucleotide was examined by
determining Kas for a group of cap analogues with
the Y138A mutant.  
Scheme 2   All used cap analogues were
triphosphates (n3)   m7GTP R1CH3 R2R3H m22,
7GTP R1CH3 R2CH3, R3H m32,2,7GTP R1CH3 R2,
R3 CH3 et7GTP R1CH2CH3 R2CH3,
R3H m7GpppG R1CH3 R2R3H R4OH BG m7GpppGm
2' R1CH3 R2R3H R4OCH3 BG m7GpppA R1CH3
R2R3H R4OH BA m7Gpppm6A R1CH3 R2R3H R4
OH Bm6A m7GpppAm2' R1CH3 R2R3H R4OCH3 B
A m7Gpppm7G R1CH3 R2R3H R4OH Bm7G et7Gpp
pG R1 CH2CH3 R2R3H R4OH BG m7GpppU R1CH3
R2R3H R4OH BU m7GpppC R1CH3 R2R3H R4
OH BC m22,7GpppG R1CH3 R2CH3,
R3H R4OH BG m32,2,7GpppG R1CH3 R2,R3H R4
OH BG A tetranucleotide mRNA fragment
m7GpppAm2'pUm2'pAm2 consists of m7GpppAm2' with
linked U and A.

Scheme 1
Results Discussion CBC significantly prefers to
bind the monomethylated dinucleotide cap analogs
comparing with the mononucleotides, except for
the compounds hipermethylated at the N2 amino
group of the 7-methylguanosine moieties. The
average binding energy of the dinucleotides is by
1 kcal/mol greater (more negative) than that for
m7GTP, which is accompanied by a drop of one
order of magnitude in terms of the association
constant values (Table, Figure2). This preference
agrees with former results of in vitro and in
vivo biological experiments (5). The titration
experiments with a tetranucleotide mRNA fragment,
m7GpppAm2pUm2pAm2 yielded slightly smaller
binding energy than that for the corresponding
control dinucleotide, m7GpppAm2. This suggests
that only two first nucleotides at the mRNA 5
terminus are responsible for the tight, specific
interaction with the CBC. However, further
nucleotides may be involved in nonspecific
contacts with the protein. The differences of ?G?
should be ascribed to the entropic costs of
ordering of the longer oligonucleotide chain upon
the binding. The purine base in the first
transcribed nucleoside is preferred. The average
energy of binding for the dinucleotides with
pyrimidine (C or U) as the second base is by 0.4
kcal/mol less than the average binding energy for
the purine (A or G) dinucleotides, and their Kas
values are 2-fold lower, respectively. m7GpppG
is bound stronger than m7GpppA unless adenosine
is additionally methylated at the amino group or
at 2'-hydroxyl. The Y138 mutant binds m7GpppG
with Kas 4-fold lower than the wild type CBC
leading to a decrease of ?G? by 0.80 0.36
kcal/mol, whereas for m7GTP the change is
negligible. A difference in a mode of m7GpppG
binding is observed while for the wild type CBC
the fitting residuals have non-random
distribution resulting from systematic deviations
(Figure 1a)), in case of the Y138A mutant the
deviations are diminished (Figure 2b)). For the
wild type CBC, these deviations appeared for the
dinucleotides containing pyrimidine as the second
base and not for the purine dinucleotides. The
modes of binding of the hipermethylated mono- and
dinucleotide cap analogs by CBC are distinctly
different. Whereas introduction of either one or
two methyl groups at the amino group of
7-methylguanosine results in a dramatic
diminution of Kas by 100-fold (decrease of ?G?
by 2.5 kcal/mol) for the dinucleotides, the Kas
values for the corresponding mononucleotides
surprisingly change only less than 2-fold. The
total fluorescence quenching pattern also reveals
the differences. It is observed only a few
percent of quenching for m32,2,7GTP while it is
up to 12 for m32,2,7GpppG. This surprising
discrepancy is the same for the Y138A mutant
(Table, Figure 2) which points to the steric
hindrance caused already by the first methylation
at the m7G amino group. This prevents the
hipermathylated cap from deep penetration into
the binding pocket.

Figure 1.
Table. Equilibrium association constants (Kas)
and binding Gibbs free energie (DG0) for various
cap analogues-CBC (wilde type) and cap-Y138A
mutant complexes at 20?C.
Figure 2. Graphical presentation of the Gibbs
free energies of association of the cap analogs
with a) wild type CBC, and b) Y138A mutant.
Conclusions The presented results together with
the structural view known from crystallography
make the first step to build molecular mechanism
of specific recognition and binding between CBC
and mRNA 5 cap. The enhanced affinity for the
dinucleotide cap analogues, especially these
containing pyrimidine as the second base, as well
as different modes of binding of mono- and
dinucleotides point at the significance of the
first transcribed mRNA nucleoside for the CBC
recognition. The results indicate the
possibility that the two-partite cap-binding
center of the CBC can bind the first and the
second nucleosides belonging to cap with some,
still unknown, level of cooperativity.
References   1. Mazza, C., Ohno, M., Segref, A.,
Mattaj, I. W., Cusack, S. (2001) Mollecular
Cell 8, 383396. 2. Mazza, C., Segref, A.,
Mattaj, I. W., Cusack, S. (2002) The EMBO
Journal 21, 55485557. 3. Calero, G.,Wilson, K.,
Ly, T., Rios-Steiner, J. R., Clardy, J.,
Cerione, R. A. (2002) Nature Struct. Biol. 9,
912917. 4. Niedzwiecka, A., Marcotrigiano, J,
Stepinski, J., Jankowska-Anyszka, M.,
Wyslouch-Cieszynska, A., Dadlez, M., Gingras,
A.-C., Mak, P., Darzynkiewicz, E., Sonenberg, N.,
Burley, S. K., and Stolarski, R. (2002) J. Mol.
Biol. 319, 615635. 5. Izaurralde, E., Lewis, J.,
McGuigan., Jankowska, M., Darzynkiewicz, E., and
Mattaj, I.W. (1995) Cell 78, 657668.
Acknowledgements This work was supported by the
State Committee for Scientific Research KBN 3
P04A 021 25, PBZ/KBN/059/T09/2001, BST 833/BF. We
are indebted to Teija Nittymaki and Harri
Lönnberg for cooperation in preparing the
tetranucleotide.