The EMBO Journal vol.10 no.9 pp.2645-2651, 1991
An intact Box C sequence in the U3 snRNA is required
for binding of fibrillarin, the protein common to the
major family of nucleolar snRNPs
Susan J.Baserga, Xiangdong W.Yang and
Department of Molecular Biophysics and Biochemistry. Howard
Hughes Medical Institute, Yale School of Medicine, 333 Cedar St,
New Haven, CT 06511, USA
Communicated by J.A.Steitz
The mammalian U3 snRNP is one member of a recently
described family ofnucleolar snRNPs which also includes
U8, U13, U14, X and Y. All of these snRNPs are
immunoprecipitable by anti-fibrillarin autoantibodies,
suggesting the existence ofa common binding site for the
34 kDa fibrillarin (Fb) protein. Two short nucleotide
sequences, called Boxes C and D, present in each ofthese
RNAs are the most likely sites for fibrillarin binding. We
have developed a HeLa in vitro assembly system for
binding offibrillarin to human U3 snRNA. Reconstitution
of the input RNA is specific in our assay since four of
the other nucleolar small RNAs (U8, U13, X and Y)
which have Boxes C and D become immunoprecipitable
by anti-fibrillarin whereas two RNAs which lack these
sequences (5S and 5.8S) do not. Deletion analyses of the
U3 snRNA demonstrate that the presence of Box C but
not Box D is required for fibrillarin binding. Moreover,
seven single or double site-specific mutations in the U3
Box C abolish binding. The role ofthe Box C-fibriliarin
interaction in the biogenesis of the Fb snRNPs
Key words: fibrillarin/nucleolus/snRNPs/U3 snRNA
U1, U2, U3, U4, U5 and U6 constitute the major small
nuclear ribonucleoprotein particles (snRNPs) in mammalian
cells because of their high abundance (0.2-1 x 106
copies/cell). Each snRNP consists of one highly structured
RNA and as many as 10 different proteins. With the
exception of U3, all of these snRNPs participate in pre-
mRNA splicing and can be found in spliceosomes in the
nucleoplasm. The spliceosomal snRNPs, except for U6, are
recognize a common epitope on several of the particle
proteins (reviewed in Luhrmann, 1988; Zieve and Sauterer,
1990). In contrast, the U3 snRNP participates in rRNA
processing (Kass et al., 1990; Savino and Gerbi, 1990) and
is located in the nucleolus (Nakamura et al., 1968; Weinberg
and Penman, 1968; Prestayko et al., 1970; Tyc and Steitz,
1989). It is immunoprecipitable byantibodies directed against
an abundant 34 kDa nucleolar protein (Lischweetal., 1985)
called fibrillarin, and not by anti-Sm sera. The U3 snRNP
therefore belongs to a distinctly different class than the
spliceosomal snRNPs by virtue of its function, subcellular
location, and protein composition.
The biogenesis of the snRNPs of the Sm class has been
well-studied. The snRNAs, except for U6, possess a short
conserved sequence [PuA(U)nGPu] which was originally
shown to be the binding site for the Sm proteins (B', B, D,
E, F, G) by RNase digestion of native snRNP particles
(Liautard et al., 1982). Specific mutations studied both in
vivo and in vitro confirmed the importance of this sequence
for binding of the Sm proteins (Hernandez and Weiner,
1986; Mattaj, 1986; Hamm et al., 1987; Surowy et al.,
1989). In addition to the common Sm proteins, each snRNP
binds specific proteins. For example, the Ul snRNP contains
three specific proteins called the 70K, A and C. The
arrangement of these proteins in the U1 RNP has been
determined using: (i) in vitro assembly of in vitro synthesized
Ul RNA with HeLa cell extracts (Patton et al., 1987; Patton
and Pederson, 1988; Lutz-Reyermuth and Keene, 1989;
Scherly et al., 1989; Surowy et al., 1989); (ii) in vitro
assembly of in vitro synthesized Ul RNA with Xenopus
oocyte extracts (Hamm et al., 1987, 1988; Scherly et al.,
1989); (iii) direct binding of Ul RNA to U1-specific proteins
synthesized in vitro (Spritz et al., 1987; Query et al., 1989;
Scherly et al., 1989; and Surowy et al., 1989); and (iv)
expression of mutated U1 genes injected into Xenopus
oocytes (Hamm et al., 1990b). Further studies of the
assembly of the Ul snRNP in Xenopus oocytes and in
mammalian cells have localized the steps in its biogenesis
to various cell compartments (Feeney et al, 1989; Fischer
and Liihrmann, 1990; Hamm et al., 1990a; Neuman de
Vegvar and Dahlberg, 1990). After transcription by RNA
polymerase II in the nucleus, Ul RNA quickly exits to the
cytoplasm where it binds pre-assembled Sm proteins and its
7-methyl guanosine cap undergoes modification to become
trimethylated (2,2,7-trimethyl guanosine). Only then is the
nascent Ul particle imported into the nucleus and its
assembly completed by binding ofthe U1-specific proteins.
Import and trimethylation are not affected by deletion of the
binding sites for the specific proteins from U 1 RNA, whereas
the Sm binding site is crucial for these processes.
In contrast to the U1 snRNP, very little is known about
the assembly of the U3 snRNP. The U3 snRNP consists of
one 217 nucleotide-long trimethyl guanosine-capped RNA
and six proteins (74, 59, 34, 30, 13 and 12.5 kDa; Parker
and Steitz, 1987). The 34 kDa protein, fibrillarin, is common
to at least five other nucleolar snRNPs (U8, U13, U14, X
and Y; Tyc and Steitz, 1989; Liu and Maxwell, 1990).
Except for fibrillarin, the protein componentsof these other
nucleolar snRNPs are not yet known. The U3, U8, U13 and
U14 RNAs possess two short nucleotide sequences called
Boxes C and D (see Figure la) which are also conserved
throughout U3 evolution (Wise and Weiner, 1980; Hughes
et al., 1987; Jeppesen et al., 1988; Tyc and Steitz, 1989).
RNAs X and Y also contain Box D and the most highly
conserved first six nucleotides of the Box C sequence
(UGAUGA and UGAAGA, respectively; K.Tyc, personal
communication). Immunoprecipitationwith anti-fibrillarin
Box C is required for fibrillarin binding to RNA
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Received on April 19, 1991