Table Of ContentMax L. Birnstiel (Ed.)
Structure and Function of
Major and Minor Small Nuclear
Ribonucleoprotein Particles
With 39 Figures
Springer-Verlag
Berlin Heidelberg New York London Paris Tokyo
Prof. Dr. MAx L. BIRNSTIEL
Institute of Molecular Pathology
Dr. Bohrgasse 9
A-1030 Wien
ISBN -13: 978-3-642-73022-1 e-ISBN -13: 978-3-642-73020-7
DOI: 10.1007/978-3-642-73020-7
Library of Congress Cataloging-in-Publication Data. Structure and function of major and minor
small nuclear ribonucleoprotein particles 1 Max L. Birnstiel (ed.). p. cm. Includes index:es. 1.
Nucleoproteins. 2. Nucleoproteins - Structure-activity relationships. I. Birnstiel, Max L., 1933-
QP625.N87S78 1988 574.87'3283-dc19 87-27690
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Preface
In the past decade a most interesting story of the role played by the small nuclear
RNPs, catalysts in RNA processing, has unfolded. Early investigations of the
structure of these particles gave rise to hypotheses of their functions. As described
in this book, these have been confirmed or are being tested by biochemical ex
perimentation and most recently by the powerful technique of genetic analysis.
Both biochemical and genetical approaches will also determine to what degree,
and how, snRNPs and their cofactors participate in the differential expression of
genes. As the details emerge and the number of snRNP species increases way
beyond the six initially identified, one feels at the threshold of even greater things
to come.
The book covers many results in one of the most rapidly expanding fields of
molecular biology. For this reason alone the specialist may detect some omissions
and shortcomings. I hope they will be few. Instead of presenting a collation of
conference reports with much overlap between them, this book has been written
specially for the purpose of surveying the literature up to early 1987 in the snRNP
field in a coherent manner, each of the seven chapters having been produced by
connoisseurs of their field. While not each and every snRNP story can be covered
in such a book, I hope that it will provide enjoyable and stimulating reading.
January 1988 MAx L. BIRNSTIEL
Contents
Small Nuclear RNAs: RNA Sequences, Structure, and Modifications
R. REDDY and H. BUSCH (With 12 Figures) ......................... 1
The Genes and Transcription of the Major Small Nuclear RNAs
J. E. DAHLBERG and E. LUND (With 1 Figure) ...................... 38
snRNP Proteins
R. LOHRMANN (With 5 Figures) 71
UsnRNP Assembly and 'fiansport
I. W. MATTA] (With 2 Figures) .................................... 100
Functions of the Abundant U-snRNPs
J. A. STEITZ, D. L. BLACK, V. GERKE, K. A. PARKER, A. KRAMER,
D. FRENDEWEY and W. KELLER (With 5 Figures) .................... 115
Structure and Function of Minor snRNPs
M. L. BIRNSTIEL and F. J. SCHAUFELE (With 6 Figures) .............. 155
Ribonuclease P as a snRNP
S. ALTMAN, H. A. GOLD and M. BARTKIEWICZ (With 4 Figures) 183
Genetic Analysis of Yeast snRNAs
C. GUTHRIE (With 4 Figures) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
Subject Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 213
Contributors
You will find the addresses at the beginning of the respective contribution
ALTMAN, S. 183 KELLER, W. 115
BARTKIEWICZ, M. 183 KRAMER, A. 115
BIRNSTIEL, M. L. 155 LUHRMANN, R. 71
BLACK, D. L. 115 LUND, E. 38
BUSCH, H. 1 MATTAJ, 1. W. 100
DAHLBERG, J.E. 38 PARKER, K. A. 115
FRENDEWEY, D. 115 REDDY, R. 1
GERKE, V. 115 SCHAUFELE, F. J. 155
GOLD, H.A. 183 STEITZ, J. A. 115
GUTHRIE, C. 196
Small Nuclear RNAs: RNA Sequences, Structure, and
Modifications
RAM REDDY and HARRIS BUSCH1
CONTENTS
Summary....................................................................... 1
1 Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2 Occurrence. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.1 Animal Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.2 Plant Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.3 Single Cell Eukaryotes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3 Diversity of Small RNAs .................................................... 5
4 Small RNAs of Normal and Thmor Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
5 Subcellular Localization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
5.1 Small RNAs in the Nucleoplasm............................................. 10
5.2 Small RNAs in the Nucleolus................................................ 10
5.3 Small RNAs in the Cytoplasm............................................... 10
5.4 Small RNAs in the Mitochondria............................................. 10
6 U-snRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
6.1 U-snRNAs of the Nucleoplasm. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
6.2 U-snRNAs of the Nucleolus................................................. 16
7 Modified Nucleotides in U-snRNAs........................................... 18
8 Comparison of U-snRNAs from Higher and Lower Eukaryotes . . . . . . . . . . . . . . . . . . . 19
9 Conserved Features in U-snRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
9.1 m3G-cap Structure........ ... ... .............. ..... .. ...... ................. 20
9.2 Primary Sequence.......................................................... 21
9.3 Conserved Secondary Structures...... ... ..... ..... .............. ..... . ....... 21
9.4 Sm-Antigen Binding Site. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
9.5 Evolution of U-snRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
10 Conclusions and Prospects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
11 Appendix. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Summary
All eukaryotic cells contain multiple small nuclear RNAs, designated U-snRNAs.
At present ten U-snRNAs have been identified; they account for about 10J0 of the
total mammalian cellular RNA. Of these, the U1 to U8 snRNAs have been exten
sively characterized. All the U-snRNAs contain a 5'-cap structure consisting of
a blocked 5'-terminal pyrophosphate linkage; except for U6 RNA, a trimethyl
guanosine-containing "cap" is the 5'-end. These U-snRNAs are confined to the
nucleus and are metabolically stable. They are synthesized by RNA polymerase
1 Baylor College of Medicine, Department of Pharmacology, Houston, Thxas 77030, USA
2 R. Reddy and H. Busch
II. U6 snRNA, which has a non-nucleotide cap structure, is synthesized by RNA
polymerase III. All the U-snRNAs are present in the cells as small ribonucleopro
tein particles (snRNPs), that are metabolically stable.
The U1, U2, U4/U6, and US snRNPs are localized in the nucleoplasm and
are involved in the maturation of pre-messenger RNAs as essential components
of the spliceosomes. The U3 and U8 snRNPs, which are localized to the
nucleolus, are implicated in processing of pre-ribosomal RNA.
The U-snRNAs have been characterized from animal cells, plant cells, smaller
eukaryotes like yeasts, slime molds, and dinoflagellates. The secondary structures
of individual U-snRNAs from distant species are virtually identical and some
regions of primary sequence are also conserved. The detailed mechanisms of in
teractions between the U-snRNPs, mRNA, and rRNA precursors are currently
under intense investigation.
In addition to the U-snRNAs, eukaryotic cells also contain multiple other
distinct small RNAs. These RNAs, found mainly in the cytoplasm and synthesiz
ed by RNA polymerase III, are involved in the protein synthesis. Although about
20 distinct small RNAs are characterized, the functions have been clearly
established for only 7SL RNA and RNase P RNA. 7SL RNA is an integral com
ponent of the signal recognition particle and RNase P RNA is part of the RNase
P complex that cleaves the 5'-ends of precursor tRNA molecules. Understanding
the functions of these cytoplasmic small RNAs is a challenge for the future.
1 Nomenclature
The U-snRNAs were designated U1, U2, U3, etc. because these RNAs were
uridylic acid-rich when compared to ribosomal or messenger RNAs (Hodnett and
Busch 1968). The term small nuclear RNA (snRNA) was introduced by Weinberg
and Penman (1968) and the term small cytoplasmic RNAs (scRNAs) was added
later (Zieve and Penman 1976). The term small nucleolar RNAs (snoRNAs) was
suggested for U-snRNAs of the nucleolus (Reddyet al. 1981 e). The U-series of
small RNAs (U1 to U3) were found to contain a cap structure on their 5'-ends
(Reddyet al. 1974; Ro-Choi et al. 1974), and all the other capped small RNAs
are added to this series. U4, US, and U6 snRNAs were designated by M. R. Lerner
and Steitz (1979), U7 snRNA by Birnstiel (Strub et al. 1984) and U8 to U10
snRNAs by Reddy et al. ( 1985 b). U 11 RNA has recently been identified by Keller
(see chapter by Birnstiel, this Vol.). Recently, Guthrie and her co-workers (Riedel
et al. 1986) have suggested that there may be more than 20 U-snRNAs in yeast,
some of which may be present in as few as 50 copies per cell. The numbers for
the U-snRNAs were designated by order of discovery and not by size, location or
abundance. Only capped small RNAs are added to the U-snRNA series.
Although the term U-snRNAs originated because of high uridylic acid content
of the original small RNAs (Hodnett and Busch 1968), some of the newer U-series
of RNAs do not contain a high proportion of uridylic acid. The 5'-cap structure
is the most definitive structural criterion for the U-snRNAs. The U-snRNAs are
usually isolated from nuclear 4-8S RNA and the fractionation and the relative
electrophoretic migration of rat U-snRNAs is shown in Fig. 1.
Small Nuclear RNAs: RNA Sequences, Structure, and Modifications 3
Fig. 1. Small RNAs of Novikoff hepatoma j 8S
cells. The nuclear 4-8S RNA of Novikoff
~_~-3.8-2
hepatoma cells was electrophoresed on a
10070 polyacrylamide gel. The RNAs were ~;;::::::::::;;:::;= -=; 7 S K
F -7SL
visualized by staining with methylene blue.
::==== ~7-2
For other details see Reddy et al. 1981 d; P
snRNA (Hotta and Stern 1981; W-RNA
U3-< 7-1
(Miller et al. 1984); and Vault RNA (Keder U2 --- -=====>-5.8S,6S
sha and Rome 1986)
U1-. -./W
U8 - -----Vault
:::= -./
~~ 5.1S
5S, P-snRNA
U5 - -----Y1
U6- Y2,4.5S1
!!!!~~>-4.5S, tcRNAs
} Pre-tRNAs
moxi-tRNAs
tRNAs
U7
>-
U10
The nomenclature of non-capped small RNAs is confusing, but the deter
mination of their functions may resolve this confusion. There are several struc
turally distinct small RNAs in the 90 nucleotide size range designated 4.5S RNA,
4.5S1 RNA, etc. Several distinct RNAs of 7S size were designated K, L, and M
by Zieve and Penman (1976). Ullu and Melli (1982) combined these two designa
tions and called these RNAs 7SL RNA, 7SK RNA, etc. The size and names of
different non-capped small RNAs reported to date are shown in Fig. 1. Renaming
these RNA species with names related to their function, for example, the 7SL
RNA to SRP-7SL RNA, SRP-7S RNA or simply SRP-RNA, may be helpful in
the future.
The small RNAs can be grouped under two classes based upon differences in
structure and function. These are: (1) capped small RNAs (snRNAs; U-snRNAs)
Table 1. Two classes of small RNAs
RNAs Capped snRNAs Non-capped
Property (U-snRNAs) small RNAs
5'-ends Usually m3GpppA; pppG; pppA
rarely m3GpppG;
U6 RNA undefined cap
3'-ends Any nucleotide Usually U; somtimes A
Modified Present Absent
nucleotides
Localization Nucleus Usually cytoplasm
Synthesized by Pol. II U6 RNA by Pol. III
Pol. III
Functions Processing of Diverse functions; major scRNAs
precursor RNAs involved in translation process
4 R. Reddy and H. Busch
and (2) non-capped small cytoplasmic RNAs (scRNAs). The properties of these
two classes of small RNAs are described in Thble 1.
2 Occurrence
2.1 Animal Cells
Early studies on rat Novikoff hepatoma cells (Hodnett and Busch 1968) and
human HeLa cells (Weinberg and Penman 1968) clearly established the existence
of distinct small RNAs in nuclei of these cells. These U-snRNAs have since been
shown to be ubiquitous and have been characterized in aves, e. g. chickens (Roop
et al. 1981), amphibians, e. g., frogs (Forbes et al. 1984), insects, e. g. fruit flies
(Mount and Steitz 1981; Myslinski et al. 1984), and many other species.
2.2 Plant Cells
Recently, several studies have been focused on U-snRNAs of plants. Skuzeski and
Jendrlsak (1985) characterized wheat U2 snRNA and found that it is highly
modified and is homologous to the rat U2 snRNA. Krol et al. (1983) used the an
ti-m3G-cap antibodies to show the presence of U1, U2, and U5 snRNAs in pea
cells. Kiss et al. (1985) showed the presence of U2, U3 and U6 snRNAs in beans.
Van Santen and Spritz (1987) characterized a bean U1 snRNA gene. From these
results, it is clear that plant cells contain the major U-snRNAs that have been
characterized in animal cells; in addition, plant cells contain several small RNAs
that have not yet been characterized (Kiss et aI. 1985). These data suggest that the
complexity of snRNAs in plant cells parallels that found in animal cells.
2.3 Single Cell Eukaryotes
Small nuclear RNAs have been reported in Amoeba proteus (Goldstein and Ko
1974), Tetrahymena (Branlant et aI. 1983), slime mold Dictyostelium (Wise and
Weiner 1981), dinoflagellates (Reddy et aI. 1983 b), malarial parasite (Francoeur
et aI. 1985) and in yeast (Wise et aI. 1983; Pikielny and Rosbash 1986; Riedel et
aI. 1986). The U5 RNA of Tetrahymena is 40070 homologous to the human U5
RNA (Branlant et aI. 1983); the U3 RNAs of slime mold and rat are 40%
homologous (Wise and Weiner 1980); yeast cells contain U2 RNA homologous
to rat U2 RNA (Ares 1986). The U2 snRNA from Trypanosomes has been charac
terized and is homologous to metazoan U2 RNA (Tschudi et aI. 1986). The
dinoflagellates contain U1 to U6 snRNAs, which are structurally homologous to
rat U-snRNAs (Reddyet aI. 1983b). Hellung-Larsen and Frederiksen (1976) have
carried out a detailed survey of small RNAs in cells from different taxonomic
levels and found small RNAs in all eukaryotic cells except in yeast. Although
more detailed characterization of snRNAs is needed for lower eukaryotes, it is
clear that structurally and functionally homologous U-snRNAs are present in
lower eukaryotes. There is no evidence, as yet, for the presence of U-snRNAs in
bacteria. .
Small Nuclear RNAs: RNA Sequences, Structure, and Modifications 5
Table 2. Copies/cell and size of U-snRNAs. The size of U-snRNAs includes the cap nucleotide; they
are from compilation of U-snRNA sequences (Reddy 1986). The copy numbers for HeLa cell U1 to
U6 snRNAs were from Weinberg and Penman 1968; sea urchin U7 RNA from Birnstiel et aI. 1985;
Novikoff hepatoma U8 to utO snRNAs are from Reddy et aI. 1985b.
RNA Chain length Copies/cell
(in thousands)
U1RNA 165 1000
U2RNA 188/189 500
U3RNA 217 200
U4RNA 143-145 200
us
RNA 116/117 200
U6RNA 106/107 200
U7RNA 57/58 30
U8RNA 140 25
U9RNA -130 >25
utORNA -60 >25
ut1 RNA -135 >25
rRNAs 5000
7SL RNA 300/301 500
Concentration oj Small RNAs in HeLA Cells. In HeLa cells, the concentration
of some of the U-snRNAs approaches the concentration of ribosomal RNAs
(Weinberg and Penman 1968). Thble 2 shows the concentration and the chain
lengths of different small RNAs in HeLa cells. The concentration of U-snRNAs
in lower eukaryotes is much less and is discussed later.
3 Diversity of Small RNAs
Using subcellular fractionation and high resolution polyacrylamide gels, the
abundant small RNAs like the U1 to U6 snRNAs and 7S RNAs were demonstrat
ed to be distinct RNAs. In 1979, Lerner and Steitz (1979) made the important
observation that some antibodies found in the sera of patients with systemic lupus
erythematosus are directed against snRNP particles. Using this sensitive im
munoprecipitation method, several new small RNAs have been identified. These
include Y-RNAs (Hendrick et al. 1981),7-2 and 8-2 RNAs (Reddyet al. 1983a).
Small RNAs found in the immunoprecipitates with different antibodies from pa
tients with autoimmune diseases are shown in Fig. 2.
This method had the advantage of detecting minor small RNAs and also
separating RNAs that comigrated using fractionation methods based on physical
properties. The abundant U5 snRNA and minor Y1 RNA comigrate on acryl
amide gels (Fig. 2); however, these RNAs were purified by immunoprecipitation
with specific antibodies. The antibodies specific to the trimethyl guanosine cap
structure have been used to detect distinct minor U-snRNAs in yeast (Riedel et
al. 1986 and 1987) and in metazoans (Reddy et al. 1985 b). The antibodies specific
to small RNPs characterized so far and the small RNAs that are found in the im
munoprecipitates are listed in Thble 3.
