Pharmac.Ther.Vol. 54, pp. 249-267,1992 Printed in Great Britain. All rightsre-~xved

0163-7258/92$15.00 © 1992PergamonPressLtd

Associate Editor: P. K. CHIANG

METHYLATED CAP STRUCTURES IN EUKARYOTIC RNAs: STRUCTURE, SYNTHESIS A N D FUNCTIONS RAM REDDY,* RAVINDER SINGHt a n d SHIGEKI SHIMBA

Department of Pharmacology, Baylor College of Medicine, Houston, TX 77030, U.S.A. Abstract--There are more than twenty capped small nuclear RNAs characterized in eukaryotic cells. All the capped RNAs appear to be involved in the processing of other nuclear premessenger or preribosomal RNAs. These RNAs contain either trimethylguanosine (TMG) cap structure or methylated y phosphate (Mppp) cap structure. The TMG capped RNAs are capped with MYG during transcription by RNA polymerase II and trimethylated further post-transcriptionally. The Mppp-capped RNAs are transcribed by RNA polymerase III and also capped post-transcriptionally. The cap structures improve the stability of the RNAs and in some cases TMG cap is required for transport of the ribonucleoproteins from cytoplasm to the nucleus. Where tested, the cap structures were not essential for their function in processing other RNAs. CONTENTS 1. Introduction 2. Diversity of Cap Structures in Nature 2.1. 2'-O-Methylations in the cap structure 3. RNA Polymerases Involved in the Synthesis of the Capped RNAs 3.1. M~G-capped RNAs 3.2. TMG-capped snRNAs 3.3. Mppp-capped snRNAs 3.4. Correlation between RNA polymerase and cap structure 3.5. Initiation nucleotide 4. Formation of Cap Structure in RNAs 4.1. M7G cap formation 4.1.1. The capping enzyme: Association of an RNA 5'-triphosphatase activity with mRNA guanylyltransferase 4.1.2. Coupling of capping and transcription initiation 4.1.3. MYG cap formation in viral RNAs 4.2. TMG cap formation 4.3. Mppp cap formation 4.3.1. Capping signal of U6 snRNA 4.3.2. Mppp cap formation can be uncoupled from transcription 4.3.3. y-Phosphate is retained during the capping of U6 snRNA 4.3.4. Factors for Mppp cap formation 5. Evolution of Cap Structures: Is MpppG/MpppA Cap Primitive? 6. Functions of Cap Structures 7. Future Prospects Acknowledgements References

250 251 251 251 251 252 252 253 253 254 254 255 255 256 256 257 257 257 257 258 259 260 262 262 262

*Corresponding author. tPresent address: Program in Molecular Medicine, University of Massachusetts, Worcester, MA 06105, U.S.A. Abbreviations--AdoMet, S-adenosylmethionine; AdoHcy, S-adenosylhomocysteine; Mppp, ?-monomethyl triphosphate; MpppG, y-monomethyl guanosine triphosphate; pol II, RNA polymerase II; pol III, RNA polymerase III; PSE, proximal sequence element; snRNA, small nuclear RNA; snoRNA, small nu¢leolar RNA; TMG, trimethylguanosine; U snRNA, uridylic acid-rich small nuclear RNA; U1 RNA, U2 RNA, etc., distinct small nuclear RNAs in the U snRNA series involved in the processing of other nuclear RNAs; B2 RNA, a capped small RNA homologous to repeated DNA in rodents; 7SK RNA, a capped small nuclear RNA of 7S size; VSV, vesicular stomatitis virus. 249

250

R. REDDY et al. 1. I N T R O D U C T I O N

RNAs in eukaryotic cells can be classified into four categories: ribosomal RNAs (rRNA), transfer RNAs (tRNAs), messenger RNAs (mRNAs), and small RNAs. rRNAs and tRNAs account for over 90% of the cellular RNA. The small RNAs account for about 1% of the total RNA in higher eukaryotes like human cells and for less than 0.1% in the case of lower eukaryotes like yeasts. Although small RNAs, like mRNAs, represent a small fraction of the cellular RNAs, they are very heterogeneous in size and abundance; in addition, some of these RNAs are also tissue specific and/or developmentally regulated. All small nuclear, including small nucleolar, RNAs (snRNA and snoRNA, respectively) are either proven or implicated in the processing of precursor mRNAs or precursor rRNAs (Steitz et al., 1988). Small RNAs are found in many subcellular compartments including nucleus, nucleolus, cytoplasm and mitochondria. In eukaryotic cells, all known mRNAs and most small RNAs characterized thus far contain a blocked 5' end known as the cap structure. The trimethylguanosine (TMG) cap structure (Fig. 1) containing the unusual 5'-5' pyrophosphate linkage between T M G and the initiation nucleotide was first observed during studies determining the complete nucleotide sequence of U1 snRNA (Reddy et al., 1974; Ro-Choi et al., 1974; reviewed in Busch et al., 1982). The MTG cap structure was identified in O

CH3 ~

/CH3

N

I

CH 3 /

I

12' 3 ' 1 2 ' 3'

5'

~t

P

t~

O

O

O

S'

N,"

I

Base I

"

, ,¢~"""-"k II , , I/OH OH ~JCH2 --O-- P--O-- P--O-- P--O--CH2 ~

'

I

-'o-

?

'

I

I

?

?

~./o,~ s't

iz

0 OCHs 1 0 = P --O--CH2

Trlmethylguanoslne

N~ Base

0 o

i

~Hs

G

H

O

O

I

,

"

H--C-O--P--O--P

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H

i

O

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--O--P--O--CH2

I

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y- Mono Methyl Phosphate

5'

I

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I

I

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jO,.._

J

3'1

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O

OH

J

O=P- O-CH2

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FIo. 1. Cap structures in U snRNAs. Top: M32"2'TG(TMG) cap structure found in eukaryotic UI-U5 and U7-UI3 snRNAs. The 2'-O-methylations occur only in higher eukaryotes, such as rat and HeLa cells, but not in amoeba or dinoflagellates. Bottom: Diagrammatic representation of the methylated 7-phosphate of the 5' nucleotide G (N1) of human U6 snRNA. The 2', 3' and the 5' represent the carbon moieties of the ribose sugar. MpppG found in U6, 7SK and B2 snRNAs.

Eukaryotic RNAs

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mRNAs (Rottman et al., 1974; Shatkin, 1976); and recently, a simple 7-monomethyl triphosphate (Mppp) cap structure (Fig. 1) has been identified in some snRNAs (Singh and Reddy, 1989). The TMG and Mppp cap structures have been demonstrated in many U snRNAs (uridylic acid-rich small nuclear RNA) from diverse organisms including human, Trypanosomas, amoeba, yeast, and plant cells. The anti-TMG and anti-v-monomethyl guanosine triphosphate (MpppG) cap antibodies immunoprecipitate snRNAs and snRNPs from diverse eukaryotic cells (Bringmann and Luhrmann, 1986; Krol et al., 1983; Montzka and Steitz, 1988; Riedel et al., 1986; Gupta et al., 1990a). There are many recent excellent reviews on the mRNA cap structures (Sonenberg, 1988; Furuichi and Shatkin, 1989). Therefore, in this review we focussed on the mechanism of formation and functions of TMG and Mppp cap structures. 2. DIVERSITY OF CAP STRUCTURES IN NATURE Different types of cap structures that are found in RNAs are summarized in Table 1. The MTGpppN cap structure is found in almost all messenger RNAs; some exceptions are known where instead of the M7GpppN, there is M2'7GpppN or TMGpppN cap structures. Certain viral RNAs have a protein covalently attached to their 5' termini (reviewed in Banerjee, 1980; Vartapetian and Bogdanov, 1987). The TMG cap structure is found in many small nuclear RNAs; in the case of human cells, U1-U5 and U7-U13 snRNAs containing the TMG cap structure have been characterized; in addition, many human small RNAs immunoprecipitable with anti-TMG antibodies are yet to be characterized. Approximately thirty small RNAs have been identified in yeast cells using the anti-TMG antibodies (Riedel et al., 1986). Human U6, 7SK, mouse B2 RNA and plant U3 snoRNA are known to contain the Mppp cap structure (Singh and Reddy, 1989; Gupta et aL, 1990a; Shumyatsky et al., 1990; Shimba et at., 1992). 2.1. 2'-O-METHYLATIONSIN THE CAP STRUCTURE The nucleotides adjacent to the cap structure are methylated to different extents. The 2'-Omethylated cap structures are referred to as 'cap 0', 'cap l' or 'cap 2', corresponding to the number of methylated sugar residues (Furuichi and Shatkin, 1989). In higher eukaryotes (e.g. HeLa cells) the M7G-capped mRNAs and TMG-capped small RNAs contain mainly cap 2 structures, whereas the RNAs in lower eukaryotes (e.g. yeast, amoeba) contain mainly cap 0. Discontinuously synthesized mRNA from Trypanosoma brucei contains 'cap 4' structure where the first four residues are 2'-O-methylated (Freistadt et al., 1988). All the available evidence indicates that these 2'-O-methylations are not coupled to transcription. In the case of rat U3 snoRNA, both cap 1and cap 2-containing RNA populations are found (Reddy et al., 1979), consistent with the notion that the 2'-O-methylations occur post-transcriptionally. The Mppp cap structures thus far characterized contain only cap 0. The function of these 2'-O-methylations adjacent to the cap structures, if any, is not known.

3. RNA POLYMERASES INVOLVED IN THE SYNTHESIS OF THE CAPPED RNAs 3.1. M7G-CAPPEDRNAs It is well documented that in eukaryotes RNA polymerase II (pol II), located in the nucleoplasm, synthesizes all premessenger RNAs. This enzyme is also responsible for the synthesis of most viral TABLE 1. Cap Structures Found in Eukaryotic RNAs Cap structure MTGpppN MZ7GpppN M2'2'7GpppN MpppN

Found in Most messenger RNAs Few messenger RNAs Many small nuclear RNAs Some messenger RNAs Some small nuclear RNAs

M, methyl; N, any nucleotide. JF~ 54/3~C

References Shatkin, 1976 Furuichi and Shatkin, 1989 Reddy and Busch, 1988 Liou and Blumenthal, 1990 Reddy and Singh, 1991

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R. REDDYet al.

RNAs in virus-infected cells. The mRNAs containing M2,7G and TMG cap structures are also synthesized by pol II.

3.2. TMG-cAPPED snRNAs Several lines of evidence suggest that pol II is responsible for the synthesis of TMG-capped U snRNAs: (1) The synthesis of these RNAs is inhibited by low concentrations of ~-amanitin in whole animals (Ro-Choi et al., 1976), cultured cells (Frederiksen et al., 1978; Chandrasekharappa et al., 1983), isolated nuclei (Roop et al., 1981; Lobo and Marzluff, 1987), cell-free extracts (Morris et al., 1986; Lund and Dahlberg, 1989; Southgate and Busslinger, 1989), and frog oocytes (Murphy et al., 1982; Mattaj and Zeller, 1983; Skuzeski et al., 1984; Reddy et al., 1987). (2) When Gram-Jensen et al. (1979) used a cell line containing an altered pol II which is 800 times more resistant towards inhibition by a-amanitin than the wild-type enzyme, the synthesis of U1, U2 and U3 snRNAs was not inhibited by high concentrations of ct-amanitin. Furthermorel synthesis of these U snRNAs is inhibited at nonpermissive temperature in the cells that contain a temperaturesensitive pol II (Hellung-Larsen et al., 1980). (3) The synthesis of U1 and U2 snRNAs is sensitive to 5-6-dichloro- l-fl-o-ribofuranosyl benzimidazole, which is a specific inhibitor of transcription by pol II at low concentrations (Hellung-Larsen et al., 1981), and the primary transcripts of U1 snRNA, like mRNAs, are co-transcriptionally capped with MTG (Eliceiri, 1980; Skuzeski et al., 1984; Mattaj, 1986). (4) Antibodies against the large subunit of the pol II inhibit the synthesis of U1 snRNA in the frog oocytes (Thompson et al., 1989). (5) Finally, RNA polymerase III (pol III) is unlikely to be involved in the synthesis of TMG-capped U snRNAs because a U cluster (AUUUUUG as Sm antigen-binding site) is present within the transcribed portion of a large number of these genes, and this results in a termination of all known pol Ill-mediated transcription. All these data show that TMG-capped snRNAs are synthesized by pol II or by an RNA polymerase closely related to pol II. Although these studies have been carried out on the synthesis of only some TMG-capped snRNAs, it is likely that other TMG-capped snRNAs are also synthesized by pol II. However, there is insufficient evidence to conclude that the pol II that synthesizes mRNAs and TMG capped snRNAs is the same. The 3' end formation in the case of U1 and U2 snRNAs is coupled to initiation from compatible U snRNA promoters, implying that the transcription complex formed on UI snRNA promoter is different from transcription complexes formed on mRNA promoters (Hernandez and Weiner, 1986; Neuman de Vegvar et al., 1986).

3.3. MPPP-CAPPED snRNAs There are four well characterized RNAs that contain the Mppp cap structure. Mammalian U6, 7SK, and B2 RNAs and the plant U3 RNA are known to contain the Mppp cap structure. There is definitive evidence to support the involvement of pol III in the synthesis of all known Mppp-capped RNAs. (1) Low concentrations of ~-amanitin, which is sufficient to inhibit the synthesis of mRNAs and TMG-capped U snRNAs, had no inhibitory effect on the synthesis of U6 RNA in frog oocytes, in vitro (Kunkel et al., 1986; Reddy et al., 1987; Krol et al., 1987), or in isolated nuclei (Kunkel et al., 1986). (2) In U6 snRNA genes, the signal for transcription termination is a T cluster (Das et al., 1988) similar to the functional termination signal in 5S RNA gene (Bogenhagen and Brown, 1981); the 3' ends of 7SK, B2 and plant U3 RNAs also consist of a cluster of four or more uridylate residues. (3) The transcription of U6 snRNA is competed by other pol III genes like 5S and tRNA genes both in vitro (Reddy et al., 1987) and m vivo (Carbon et al., 1987). (4) The U6 snRNA associates with La antigen (Rinke and Steitz, 1985; Reddy et al., 1987) which may be a pol III transcription termination factor (Gottlieb and Steitz, 1989). (5) A mutant yeast strain with temperature-sensitive defect in the large subunit of pol III, which results in a defective transcription of tRNA and 5S RNA genes, is also defective in U6 snRNA transcription (Moenne et al., 1990). (6) Tagetitoxin, a specific inhibitor of transcription by pol III at low concentrations, inhibits the synthesis of U6 snRNA (Steinberg et al., 1990). All these data show that U6 snRNA genes are transcribed by pol III. The involvement of pol III in the synthesis of

Eukaryotic RNAs

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7SK RNA is well documented (Zieve et al., 1977; Murphy et al., 1986, 1987; Kruger and Benecke, 1987). The mouse B2 RNA (Kramerov et al., 1985) and U3 snoRNA in higher plants are also synthesized by pol III (Kiss and Solymosy, 1990; Kiss et al., 1991).

3.4. CORRELATIONBETWEEN RNA POLYMERASEAND THE CAP STRUCTURE

All the M7G and TMG-capped RNAs are synthesized by pol II. The U3 snoRNA from a variety of sources including human, rat, mouse, Drosophila, Xenopus, Dictyostelium and yeast cells contains the TMG cap structure and where studied, is synthesized by pol II. Surprisingly, the U3 snoRNA from higher plant (pea) cells was not precipitable with anti-TMG antibodies (Krol et al., 1983); however, U3 snoRNA did contain a blocked 5' end (Kiss et al., 1991). Consistent with these observations, tomato U3 snoRNA genes were transcribed by pol III (Kiss and Solymosy, 1990). All these observations suggested a Mppp cap structure in plant U3 snoRNA. Our recent data confirm this speculation and cowpea U3 snoRNA was found to contain a MpppA cap structure (Shimba et al., 1992). Therefore, the U3 snoRNA contains different cap structures in different species. This appears to be true for U6 snRNA also. Although U6 snRNA in plant and animal cells is Mppp-capped, in amoeba (Physarum) it is TMG-capped (Adams et al., 1987). These data again show that the type of cap structure that any particular snRNA has is not universal and differs in different species. Whether a particular RNA contains Mppp or TMG cap structure appears to be related to the type of RNA polymerase that transcribes the genes. The 5' flanking sequences of vertebrate U1, U2 or U6 snRNA genes are capable of directing transcription, indicating that the promoters for these U snRNA genes may be exclusively in the 5' flanking region. When one compares the promoters of vertebrate U1 and U2 genes with those of the U6 genes, the main difference is the presence ofa TATA box centered around -30 in the U6 genes and its absence in U1 and U2 snRNA genes. Therefore, it should be possible to convert the U1, U2 (pol II) promoters into U6 (pol III) promoters and vice versa. This has been successfully accomplished in the case of Xenopus and human U snRNA genes (Mattaj et al.,1988; Lobo and Hernandez, 1989; Lobo et al., 1990). Alteration in the -30 TATA box in the human U6 gene results in transcription by pol II and an introduction of a TATA box into the -30 region in the human U2 gene results in transcription by pol III (Lobo and Hernandez, 1989). In the case of plants, genes for TMG-capped U2, U4 and U5 snRNAs are transcribed by pol II, and genes for MpppG-capped U6 snRNA are transcribed by pol III. Both classes of genes require only proximal sequence element (PSE) and TATA elements which are interchangeable between these two classes of genes. The spacing between these two cis-acting elements appears to be a major factor in determining which polymerase is employed. Introduction of a 10 base pairs (one helical turn) DNA between the PSE and the TATA box of Arabidopsis U6 gene results in a transcription by pol II. Conversely, deletion of 10 base pairs between the TATA box and the PSE sequence motif of Arabidopsis U2 gene results in transcription by pol III (Waibel et al., 1990). Plant U3 snoRNA transcribed by pol III contains Mppp cap structure (Shimba et al., 1992), and when transcribed by pol II contains TMG cap structure (Kiss et al., 1991) showing that the type of cap structure on an RNA is dependent on the RNA polymerase transcribing the snRNA gene. All the available data are consistent with the notion that TMG-capped U snRNAs are pol II products and MpppG/A-capped snRNAs are pol III products. In fact, it is possible that some U snRNA promoters, such as the Xenopus U6 snRNA gene promoter, may have dual polymerase specificity. Microinjected Xenopus U6 snRNA genes apparently are transcribed in frog oocytes by both pol II and pol III (Mattaj et al., 1988).

3.5. INITIATIONNUCLEOTIDE Most U snRNAs, thus far characterized, initiate with a purine nucleotide. In this respect, the synthesis of U snRNAs is similar to that of most mRNAs, tRNAs and rRNAs which also initiate with a purine nucleotide. The TMG-capped U RNAs, in general, initiate with adenosine; the only exceptions known are pea U5 and some yeast snRNAs, which initiate with guanosine (Krol et al.,

254

R. REDDYet al.

1983; Parker et al., 1988). A consensus sequence 5' YYCAYYYY 3' was observed surrounding the cap site of mRNAs, wherein A is the initiation nucleotide (Corden et al., 1980). A pyrimidine-rich, conserved consensus sequence around the initiation site is also observed for the TMG-capped U snRNA genes. Although fewer than twenty genes have been characterized for U6, 7SK and B2 snRNAs, a similar consensus (YYYGTNYT) is observed surrounding the initiation site. The sequences around the initiation nucleotide were found to be very important for both accuracy of initiation and the efficiency of transcription of Xenopus U6 snRNA gene (Mattaj et al., 1988). The initiation nucleotide does not appear to be critical in capping since T M G as well as MPPP capped RNAs are known to initiate with A or G (Reddy and Singh, 1991). In addition, U3 and U6 snRNAs where the initiation nucleotide was changed from G--+A or from A--*G were accurately capped in vitro as well as in vivo (Shimba et al., 1992). These data indicate that capping machinery functions normally regardless of the initiation nucleotide.

4. F O R M A T I O N OF CAP STRUCTURE IN RNAs 4.1. M7G CAP FORMATION In eukaryotes, pol II transcripts (mRNAs and some snRNAs) are co-transcriptionally capped, whereas transcripts synthesized by pol I or pol III are not capped on their 5' ends. Although in cells, the m R N A capping enzyme caps exclusively the pol II-transcribed RNAs, the purified capping enzyme (GTase) has not been shown to exhibit sequence specificity, at least qualitatively, because RNAs from different sources and even homopolyribonucleotides containing 5' di- or tri-phosphates are capped. Nonetheless, the element, if any, that confers specificity exclusively for pol II transcripts has not been characterized. Thus, it has been argued that the m R N A capping enzyme by virtue of its association with the transcription complex caps all the pol II transcripts regardless of their sequence (Moss, 1984; Emerson et al., 1985; Mizumoto and Kaziro, 1987; Guthrie and Patterson, 1988). However, it is not known at the moment that all RNAs in a mixture will be capped with equal efficiency. Cap formation is catalyzed by a series of enzymes (e.g. m R N A guanylyltransferase, mRNA (guanine-7-)methyltransferase and m R N A (nucleoside-2'-O)-methyltransferase). As an intermediate during capping, G M P is covalently attached to c-lysine by a phosphoamide linkage rather than a phosphoester linkage (Mizumoto et al., 1982). Formation of complete cap structure involves

a) p p p N I p N 2 p - a') p N l p N 2 p - -

c(13"t

~ ~

ppNlPN2p-ppNlpN2p--

I~'c('

b) Gppp + p p N l p N 2 p - -

+ Pi + ADP

(xl3'a' ~

c) AdoMet + G p p p N l p N 2 P - - d) AdoMet + M T G p p p N l p N 2 - -

or

13~,

G p P p N l p N 2 p - - + PP| M7GpppNlpN2p--

+ AdoHcy

MTGpppN1 mpN2p""

+AdoHcy

e) If Nlm = Am : AdoMet + M r G p p p A m p N 2 P ' -

,~ M7GpppSmAmpN2p---

f) AdoMet + M T G p p p N l m p N 2 - -

,~ MTGpppNlmpN2mP---

+ AdoHcy

+ AdoHcy

FIG. 2. Summary of a set of reactions involved in the capping of eukaryotic mRNAs. This figure shows the sequence of reactions during the capping of mRNAs. The enzymes involved are: (a) RNA triphosphatase; (a') RNA 5'-monophosphate phosphokinase; (b) mRNA guanylyltransferase; (c) 7-methylguanosine methyltransferase; (d) and (0 2'-O-methyltransferase; and (e) 6-methyl-(2'-O-methyladenosine) transferase (modified from Perry, 1981). In (b) Gppp represents guanosine-5'-triphosphate and is shown as Gppp for clarity to show the source of various phosphates in the cap structure (see Fig. 3).

Eukaryotic RNAs

255

the following steps: Steps (a) through (e) occur in the nucleus during or immediately after transcription. Reaction (f) is cytoplasmic and occurs after the mRNA has been incorporated into the polyribosomes (Fig. 2; Perry and Kelley, 1976; Perry, 1981). 4.1.1. The Capping Enzyme: Association of an R N A 5"-Triphosphatase Activity with m R N A Guanylyltransferase The mRNA capping enzymes have been isolated and characterized from HeLa cells (Ensinger and Moss, 1976; Wei and Moss, 1975; Keith et al., 1978; Venkatesan et al., 1980; Wang et al., 1982; Shuman, 1982; Langberg and Moss, 1981), rat liver (Mizumoto and Lipmann, 1978, 1979; Mizumoto et al., 1982; Yagi et al., 1983), Artemia salina (Toyama et al., 1983; Yagi et al., 1984), calf thymus (Nishikawa and Chambon, 1982), wheat germ (Toyama et al., 1983; Keith et al., 1982; Locht and Delcour, 1985), Neurospora crassa (Germershausen et al., 1978), and yeast (Itoh et al., 1982, 1984a,b; Locht et al., 1983; Wang and Shatkin, 1984; Locht and Delcour, 1985). From rat liver, both guanylyltransferase and RNA 5'-triphosphatase activities are in the same polypeptide of 69 kDa (Mizumoto and Lipmann, 1979; Mizumoto and Kaziro, 1987). In HeLa cells, these activities may be present in separate polypeptides (Venkatesan et al., 1980). Vaccinia virus contains the complex of 130 kDa containing two subunits ~ and /~ (1:1 ratio) having three enzymatic activities, i.e. mRNA guanylyltransferase, mRNA (guanine-7-)methyltransferase, and RNA 5'triphosphatase. The 95 kDa subunit of the vaccinia virus capping enzyme is known to be involved in the transguanylylation reaction. Cellular capping enzymes possess no (guanine-7-)methyltransferase activity; however, mRNA guanylyltransferase and RNA 5'-triphosphatase activities are associated physically. Enzymes from mammalian and A. salina consist of a single polypeptide (70 kDa), while in yeast, Saccharomyces cerevisiae, the enzyme is composed of two polypeptides: 80 kDa RNA 5'-triphosphatase and 52 kDa mRNA guanylyltransferase. These are encoded by two separate genes. A comparison of genes between yeast and animal cells suggests that gene fusion may have occurred during evolution (Mizumoto and Kaziro, 1987). 4.1.2. Coupling of Capping and Transcription Initiation In the case of cytoplasmic polyhedrosis virus, transcription is greatly stimulated by S-adenosylmethionine (AdoMet) and S-adenosyl-homocysteine (AdoHcy). In the presence of AdoMet, methylated cap was formed while in the presence of AdoHcy, the cap remained unmethylated. These results led Furuichi (1974, 1978) to conclude that capping is an obligate requirement for the transcription of cytoplasmic polyhederosis virus. It has been suggested that the presence of AdoMet or a similar molecule at the active site of the methyltransferase may induce a conformational change in the enzyme that is communicated to the RNA polymerase thereby allowing transcription initiation. Using an in vitro system, capping and methylation in vesicular stomatitis virus (VSV) also have been shown to be tightly linked to mRNA synthesis (Abraham and Banerjee, 1976). However, capping may not be absolutely necessary for transcription as evidenced by the following observations. The in vitro transcription from adenovirus 2 late promoter under UTP-limiting conditions produced almost all the ~-amanitin-sensitive RNAs that were shorter than 20 nucleotides. These RNAs were neither capped nor 2'-O-methylated (Coppola et al., 1983). In the case of Chinese hamster ovary cells, brief exposure to methyl-labeled methionine resulted in the appearance of label in the cap suggesting that the addition of cap on snRNAs occurs at a very early stage of RNA synthesis (Salditt-Georgieff et al., 1980). Fifty nucleotide-long nascent RNA transcripts synthesized from the adenovirus major late promoter in vitro were capped while RNAs smaller than 20 nucleotides remained uncapped. These data suggest that capping occurs when RNA chains elongate between 20 to 50 nucleotides (Coppola et al., 1983; Jove and Manley, 1984). AdoHcy inhibited transcription initiation by pol II but not by pol III, suggesting a close involvement of cap methyltransferase in transcription initiation by pol II; however AdoHcy did not inhibit the transcription by pol II purified from HeLa cells (Jove and Manley, 1982).

256

R. REDDYet al.

These results show that the formation of 5'-cap structure is coupled to transcription initiation. According to one model postulated to account for the available data, AdoHcy may interact with a component of the transcription initiation complex, probably with methyltransferase. This interaction then induces a conformational change that disrupts transcription initiation, probably by dissociating the complex. Over 95% of the transcripts synthesized by heterologous T7 RNA polymerase in mammalian cells remain uncapped, presumably because of the inability of the capping machinery to interact with heterologous T7 polymerase (Fuerst and Moss, 1989). Taken together these results suggest that transcription and capping of mRNAs is tightly coupled.

4.1.3. M7G Cap Formation in Viral R N A s With respect to reo, cytoplasmic polyhedrosis, and vaccinia viruses, the cap structures are formed by the transfer of the GMP moiety of GTP to the 5'-diphosphate end of the acceptor RNA. In contrast to the above viruses, it appears that during the capping of VSV there is a transfer of GDP moiety of GTP to the 5'-monophosphate end of RNA (Abraham et al., 1975). In the case of VSV, 2'-O-methylation precedes the guanosine-7-methylation (Testa and Banerjee, 1977).

4.2. TMG CAP FORMATION In TMG-capped snRNAs, the MTG cap is added co-transcriptionally in the nucleus (Eliceiri, 1980; Skuzeski et al., 1984), with the subsequent conversion of M7GpppA cap to TMGpppA cap occurring post-transcriptionally in the cytoplasm. In the case of Xenopus U2 snRNA, this trimethylation requires the presence of an Sm antigen-binding site. The Sin-binding site directs the formation of the TMG cap independent of its distance from the 5' end (Mattaj, 1986). Additionally, the Sin-binding site is present at different positions in different U snRNAs with respect to their 5' end (Brunel et al., 1985). The Sm-binding site is a highly conserved sequence with a consensus, PuAU(3_4)NUGPu for metazoan U snRNAs, while AU(54)GPu for yeast U snRNAs. However, despite this high degree of conservation, it is remarkably tolerant to mutations (Jones and Guthrie, 1990). Only a subset of the TMG-capped snRNAs is found associated with the Sm antigen. Human U3, U8, and U13 snoRNAs do not belong to the Sm class of snRNAs, although they contain the TMG cap structure. Therefore, there must be alternate signal(s) in these snRNAs which dictate conversion of MTG cap to TMG cap. A 34 kDa protein (fibrillarin) binds to all these three nucleolar small RNAs and it is suggested that fibrillarin-bound snRNAs might be recognized as templates by the cap methylating enzymes. Baserga et al. (1992) have recently shown that in Xenopus oocytes U3 snoRNA, like U1 and U2 snRNAs, moves transiently to the cytoplasm where its MTG cap is converted to TMG cap structure. The signal(s) in U3 snoRNA important for cap hypermethylation differs from those found in spliceosomal RNPs U1, U2, U4 and U5. These investigators also demonstrated that integrity of the T-proximal part of U3 snoRNA is essential for both TMG cap formation and import to the nucleus. While the formation of most MTG cap structures is tightly coupled to transcription by pol II, it appears that the formation of the cap structure in some Trypanosomal U snRNAs is not coupled to transcription and the capping may occur post-transcriptionally; in T. brucei, U2, U3 and the splice leader RNAs are capped post-transcriptionally in vitro (Zwierzynski and Buck, 1990). In addition, certain viral pol II transcripts lack cap structures (Sonenberg and Pelletier, 1989). These results show that in some instances MTG cap formation is not coupled to transcription by pol II. In addition to the MTG capped mRNA, some mRNAs were recently found to contain TMG cap structure. These RNAs are formed as a result of trans-splicing of a TMG cap-containing common leader sequence to a variety of recipient mRNAs and the TMG cap structure is retained on the mRNAs (Liou and Blumenthal, 1990; Doren and Hirsh, 1990). These TMG capped mRNAs are found in the cytoplasm associated with polysomes suggesting that these TMG capped mRNAs are functional mRNAs (Liou and Blumenthal, 1990). In the case of influenza virus, the 5' ends of cellular mRNAs containing the 5' cap structure serve as primers for viral mRNA synthesis; thus,

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the 5' ends of these viral mRNAs, including the 5' cap structures, are derived from cellular mRNAs (Ulmanen et al., 1983). 4.3. MPPP CAP FORMATION All the known Mppp cap-containing RNAs are transcribed by pol III. The capping of mammalian U6 snRNA in vitro is dependent on specific RNA sequence since the transcripts lacking sequences corresponding to the U6 snRNA do not get capped. For the purpose of capping, transcripts containing nucleotides 1-25 of U6 snRNA are as good substrates as the full-length U6 snRNA (Singh et al., 1990). These data indicate that the information for the formation of Mppp cap in these RNAs resides within the RNAs. This is in contrast to the capping of most pol II transcripts where capping does not depend on a specific RNA sequence. 4.3.1. Capping Signal of U6 snRNA The capping determinant of U6 snRNA is a bipartite element consisting of an intact 5' stem-loop followed by an AUAUAC sequence. Wild-type capping efficiency requires three elements in a particular context: an intact stem-loop, AUAUAC sequence immediately after this stem-loop, and pppG in close proximity of the stem-loop. This AUAUAC sequence, when present following a synthetic stem-loop, generated from vector polylinker sequence, is sufficient to convert a noncapped heterologous transcript into a capped transcript. This suggests that the stem-loop plays largely a structural role. The capping determinant in U6 snRNA shows rather stringent requirement with respect to its distance from the initiation nucleotide. This is in contrast to Sm binding-site-dependent trimethylation of MTG cap of U2 snRNA in which distance between the Sm binding-site and the cap site is variable (Mattaj, 1986). The Sm-binding site is present at different places with respect to the initiation nucleotide in different TMG-capped snRNAs (Liautard et al., 1982; Brunel et al., 1985). A comparison of the available U6 snRNA sequences shows that a stem-loop is present near the 5' end; this possibly reflects that insertions before the stem-loop are selected against during evolution to position the initiation nucleotide very close to the capping determinant. Sequences corresponding to nucleotides 1-45 of tomato U3 snRNA are capable of directing accurate cap formation in vitro (Shimba et al., 1992). Similarly, it has been found that the capping of 7SK snRNA also requires initial 29 nucleotides at its 5' end, and deletion of sequences at the 5' end abolishes capping (unpublished results). Together, these results suggest that the capping signal in these 3 Mppp-capped RNAs is at their 5' ends. 4.3.2. Mppp Cap Formation can be Uncoupled from Transcription Since the transcripts lacking initial 25 nucleotides of U6 snRNA (synthesized under U6 promoter) remained uncapped, it would be reasonable to argue that capping of U6 snRNA is not inherent to the U6 snRNA gene transcription complex. U6, 7SK, B2 and U3 RNAs having ppp on their 5'ends are accurately capped to Mppp when incubated with the HeLa extract. This post-transcriptional capping is dependent on the presence of capping determinants in these snRNAs. These data indicate that the capping and transcription of Mppp capped snRNAs can be uncoupled. This is in contrast to the capping of most mRNAs or other TMG-capped snRNAs in which capping is coupled to transcription. 4.3.3. 7-Phosphate is Retained During the Capping of U6 snRNA During the mRNA cap formation, the ~-phosphate of the mRNA is removed and does not appear in the mature mRNA (see Fig. 3). In contrast, the ~-phosphate of U6 snRNA is not removed during cap formation. Therefore, the mechanism of this U6 cap formation is very different from the mechanism that has been well characterized for mRNAs (reviewed in Banerjee, 1980). In addition to the requirement for the capping signal present within the U6 snRNA, the capping

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mRNAs, U1 - U5 snRNAs, U7 - U13 snRNAs ~13 "t ~" 13' ~' Gppp +*p p pN-~ PPi +*PI

U6, 7SK, B2 etc. AdoMet +*p

p~

AdoHcy~ 2' 13~ M pppti

FIG. 3. A comparison of the mechanism of capping of Mppp-capped U6 and 7SK snRNAs with that of mRNAs and TMG-capped U1 and U2 snRNAs. The mechanism of capping of mRNAs and U1-U5 snRNAs is shown in the left panel (see Banerjee, 1980; Perry, 1981), while the mechanism of capping of U6 and 7SK snRNAs is shown in the right panel a, fl, and 7 indicate the phosphate residues of the initiation nucleotide or the GTP used as a donor of the GMP during the capping reaction. AdoMet is the donor of the methyl group, while AdoHcy is the product of the reaction, riP-labeled 7-phosphate is marked by an asterisk (*).

machinery methylated only the 7-phosphate, although U6 snRNAs with pG and ppG on their 5' ends were also present in the reaction mixture. This suggests that the y-phosphate has to be in the right position with reference to the capping signal. Also, whenever the pppG was moved away from the capping signal, the efficiency of capping was < 1%. These data suggest that the only requirements in the U6 snRNA substrate for the capping are the capping signal and the ~,-phosphate in the right position (Singh et al., 1990; Gupta et al., 1990b). Where in the cell does methylation of Mppp cap occur? The capping of other U snRNAs takes place in two stages: the addition of MTG is coupled to transcription in the nucleus (Eliceiri, 1980; Skuzeski et al., 1984) and the trimethylation of the M7G cap is cytoplasmic (Mattaj, 1986). It is not clear as to where the Mppp cap is formed. U6 snRNA, synthesized in or injected into the oocyte nucleus, does not get transported from nucleus to the cytoplasm (Hamm et al., 1990). Approximately, 50% of the U6 snRNA synthesized in the frog oocytes is found to be capped. These data strongly suggest that capping occurs in the nucleus. When U6 snRNA synthesized by T7 RNA polymerase is injected into enucleated frog oocytes, a low level of accurate capping of U6 snRNA is observed (unpublished results), suggesting that oocyte cytoplasm has the required capping machinery. The biosynthesis and the transport pathways of capped small RNAs is shown in Fig. 4.

4.3.4. Factors f o r M p p p Cap Formation Many DNA sequence-dependent methyltransferases have been purified and characterized (Holliday, 1989; Szyf et al., 1989; reviewed in Yuan and Hamilton, 1984). Since most cellular RNAs, including ribosomal, transfer, and messenger RNAs, are methylated post-transcriptionally (Perry, 1981), RNA sequence-dependent methyltransferases must exist in the cells. Recently, accurate RNA sequence-dependent methylation of internal adenosine residues in messenger RNA has been reported (Narayan and Rottman, 1988; Harper et al., 1990); however, all the known methyltransferases modify either the base or the sugar moieties in nucleic acids. Formation of the Mppp cap structure is the first known instance of enzymatic modification of phosphate residue by an R N A sequence-dependent methyltransferase. Although the DNA and RNA methylation reactions involving AdoMet characterized earlier do not require magnesium ions, some methylation reactions are stimulated in the presence of Mg 2+ ions (Martin and Moss, 1975; Yuan and Hamilton, 1984). The methylation reaction involved in the capping of U6 snRNA requires magnesium ions, and there is no detectable capping with HeLa cell extract depleted of Mg 2÷ ions. Since the stem-loop structure in U6 snRNA is required for the U6 snRNA cap formation (Singh et al., 1990), it is likely that the Mg ~+ ions are necessary to facilitate U6 snRNA in acquiring the appropriate secondary structure required to direct the cap formation. It is also possible that the U6 snRNA sequence-dependent methyltransferase is

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UlsnRNA pppApN--AuuuuuG--

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U6 snRNA

ppApN---AUUUUUG-GpppApN--AUUUUUG~

\

M7GpppAmpN~AUUUUUG~

MpppG~

. ~ ~

NUCLEUS CYTOPLASM Sm

M7GpppAmpN~AUUUUUG~

Sm

M7GpppAmpN m--AUUUUUG-~.5 Sm M2,2,7Gp;pAmpNm_AU UUU~IIBI~G__ U FIG. 4. Mechanism of snRNA cap formation. Schematic representation of the cellular events leading to the formation of snRNA cap structures. TMG-capped snRNAs (e.g. U1 snRNA): (1) triphosphatase; (2) guanylyltransferase requiring GTP; (3 and 4) methyltransferase; and (5) Sin-dependent methyltransferase. MpppG-capped snRNAs (e.g. U6 snRNA): (1) RNA sequence-dependent methyltransferase. The methyltransferases utilize AdoMet as the methyl donor. different from other methyltransferases in its requirement for magnesium ions. All the available data on the Mppp cap formation are obtained with U6 snRNA as the substrate. Further studies are needed to see whether the same mechanism is involved in the capping of other Mppp capped RNAs.

5. EVOLUTION OF CAP STRUCTURES: IS MpppG/MpppA CAP PRIMITIVE? The mechanism of formation of different cap structures is fundamentally different (see Banerjee, 1980; Reddy and Busch, 1988; Singh et al., 1990). The formation of cap structure on pol II transcripts requires the interaction of the capping machinery with the transcription complex. On the other hand, capping of U6 snRNA is post-transcriptional. During molecular evolution, it is believed that in the primitive RNA world various chemical reactions, e.g. the processing of group 1 introns, including cleavage and ligation, the RNA replication etc., were catalyzed by the RNA itself. Subsequently, there was a transfer of more and more information from RNA to proteins in the so-called RNA-protein world in which information was present both in the RNA and the protein, e.g. translation of proteins, splicing of pre-mRNAs etc., such that each alone (RNA or protein) was not fully functional. From this, it has been convincingly argued that self-splicing of group 1 introns was a more primitive reaction from which the more elaborate reactions, e.g. the splicing of group II introns or pre-mRNA splicing, arose (Cech, 1989, 1990). As the reaction became complex, probably to insure the precision in splicing, more and more information was transferred from the substrate RNA to the trans-acting factors. Finally, all the information was transferred to the proteins as a number of biological reactions are known to be catalyzed by proteins only.

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Autocatalytic RNA

Evolution of capping machinery

?

RNA + Proteins (Ribonucleoprotein)

MpppG/A Pol III; RNA Sequence-dependent U6, 7SK, B2, etc.

Proteins

MTGpppN Pol II;RNA Sequence-independent Most mRNAs, TMG capped snRNAs

FIG. 5. Evolution of RNA capping machinery. The evolution of enzyme is shown on the left side of the figure. A comparison of the evolution of the RNA capping machinery is illustrated on the right half of the figure. ? represents a hypothetical ancestral RNA capable of Mppp cap formation without the participation of protein component.

The capping of Mppp capped RNAs appears to be an evolutionary relic of the RNA-protein world, while the capping of pol II transcripts appears to be a reaction of a more recent protein world. In the capping of U6 snRNA, specific U6 snRNA sequence and a methyltransferase are required, while during the capping of pol II transcripts there is no qualitative information within the RNA. Therefore, it can be argued that the formation of Mppp cap is more primitive compared to the formation of m R N A cap structure (Fig. 5). This is further supported by the simple nature of the U6 snRNA cap structure itself. If so, since methyltransferases are common to both prokaryotic and eukaryotic systems and AdoMet is a ubiquitous methyl group donor, it would not be surprising if Mppp cap containing RNAs are found in lower organisms like bacteria. If this hypothesis is true, one would expect Mppp cap-containing RNA genes to be transcribed by pol III in lower eukaryotes, and as evolution progressed, by pol II in higher eukaryotes. However, results available in the case of U3 RNA genes show that U3 snoRNA genes evolved from pol II in yeast to pol III in higher plants (Kiss et al., 1991). 6. F U N C T I O N S OF CAP S T R U C T U R E S The RNA cap structures, widespread in eukaryotic cells, are known to play diverse roles. Most of the work on the functions of cap structures is focussed on the MTG cap structure in mRNAs. Some of the known functions of different cap structures are summarized in Table 2. The precise function(s) of T M G cap of U1-U5 and U7-U13 snRNA and of Mppp cap of U6 and 7SK snRNAs, as well as several other RNAs, is still not clear. These cap structures appear to have roles in RNA stability, transport from cytoplasm to nucleus, and/or in other RNA function(s) yet to be identified. Using the frog oocyte system, laboratories of Mattaj and Luhrmann independently showed that the T M G cap in the case of U1 and U2 snRNAs is required for transport of these RNPs from cytoplasm to the nucleus (Fischer and Luhrmann, 1990; Hamm et al., 1990). U1 snRNA with M7G or M2'7G or MTA caps were not transported. Microinjection of anti-TMG antibodies inhibited nuclear transport of U1 snRNPs. Nuclear transport of U1 snRNA was competitively inhibited by the co-injected T M G p p p G dinucleotide, indicating that factor(s) essential for the transport of U1 snRNP can be bound to this cap dinucleotide. Oxidation of the 5' terminal ribose ring of the T M G cap also prevented transport, indicating that the transport machinery requires not only methyl groups but also the intact sugar moiety (Fischer and Luhrmann, 1990). In contrast to the results obtained with U1 snRNA where T M G cap was essential for targeting U1 snRNA into the nucleus (Fischer and Luhrmann, 1990; Hamm et al., 1990), U4 and U5

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TABLE2. Functions o f Various Cap Structures A. M7G cap structure 1. Transcription initiation 2. Generation of capped primers for influenza viral mRNA synthesis 3. Pre-mRNA splicing 4. 3'-End formation of histone mRNA 5. mRNA stability by protecting against 5' exonucleolytic degradation 6. Translation initiation by allowing ribosome binding mediated via cap binding protein 7. Role in trans-splicing B. TMG cap structure 1. Increases the RNA stability 2. Targets U1 and U2 snRNPs to nucleus C. MpppG structure 1. Increases the RNA stability

Shatkin, 1976; Furuichi, 1978; Ulmanen et al., 1983 Konarska et al., 1984; Edery and Sonenberg, 1985; Inouye et al., 1989 Georgiev et al., 1984; Hart et al., 1985 reviewed in Banerjee, 1980 Shatkin, 1985; Furuichi et al., 1977 reviewed in Banerjee, 1980 Shatkin, 1985; Furuichi et al., 1977 Ullu and Tschudi, 1991 Hammet al., 1990 Hamm et al., 1990; Fischer and Luhrmann, 1990; Fischer et al., 1991 Hamm et al., 1990

snRNAs showed a much less stringent requirement for the TMG cap structure. The Mppp cap structure in U6 snRNA was shown not to have a role in nuclear targeting (Fischer et al., 1991). The capped U1 and U2 snRNAs are more stable than the noncapped RNAs, indicating that the TMG cap increases the stability of these snRNAs (Kleinschmidt and Pederson, 1990; Harem et al., 1990) and this may hold true for other TMG cap-containing U snRNAs as well. In the case of U2 snRNA, the TMG cap does not appear to be absolutely required for 3' end processing (Hernandez and Weiner, 1986), or small nuclear ribonucleoprotein assembly (Kleinschmidt and Pederson, 1990). The nuclear targeting of U6 snRNA requires the AUAUAC sequence (nucleotides 20-25) (Hamm and Mattaj, 1989; Hamm et al., 1990), and the AUAUAC sequence is essential for the capping of U6 snRNA (Singh et al., 1990). The nuclear targeting of U1 snRNA requires both the TMG cap and binding of common U snRNP protein (Sm-protein). However, the interaction between TMG cap structure and Sm-protein is not essential, since the TMG cap can functionally substitute the Mppp cap of U6 snRNA for the nuclear targeting of U6 RNA as long as the AUAUAC sequence is present (Hamm et al., 1990). These data initially suggested that the two cap structures may be functionally equivalent, as are the Sm-binding site in U1 snRNA and the AUAUAC sequence in U6 snRNA. However, subsequent studies showed that the Mppp cap on UI RNA cannot substitute for TMG cap in the transport from cytoplasm to nucleus, and Mppp cap was found to have no role in the transport of U6 RNA from cytoplasm to the nucleus (Fischer et al., 1991). Wheat germ agglutinin, which prevents the import of many proteins from cytoplasm to the nucleus, inhibits the import of U6 snRNA, but not the import of U1 to U5 snRNAs. TMGpppG cap analog can also inhibit the import of TMG-capped UI or U5 snRNAs, but not of U6 snRNA or a karyophilic protein (Fischer et al., 1991; Michaud and Goldfarb, 1991). These data indicate that TMG capped snRNAs and U6 snRNA enter the nucleus by different pathways. The pathway used by the U6 snRNA is similar or identical to that used by karyophilic proteins. The methylation of the y-phosphate may protect U6 RNA from exonucleolytic degradation. In fact, while methylguanosine cap structures can be cleaved from capped RNAs by venom phosphodiesterase, the U6 cap is resistant (Epstein et al., 1980). Recently, one U4/U6 snRNP protein has been characterized (Baroques and Abelson, 1989), and SP6-transcribed U6 snRNA has been shown to reconstitute into snRNP particles and incorporate into spliceosomes (Pikielny et al., 1989). In these in vitro splicing systems, deletion of the 5' end of U6 snRNA that encompasses the capping determinant (nucleotides 1-25) did not affect U4/U6 snRNP assembly or spliceosome assembly (Bindereif et al., 1990). These data indicate that the U6 snRNA cap structure may not play a direct role in the assembly of U4/U6 snRNP or of the spliceosome. The U3 snoRNA from

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the animal cells contains a T M G cap and an M p p p A cap in higher plant cells. In transfected plant protoplasts, U3 s n o R N A accumulates in the nucleus and forms ~ 15S R N P particles, irrespective of whether it is transcribed by pol II ( T M G - c a p p e d R N A ) or pol III (MpppA-capped RNA) (Kiss et al., 1991). Interestingly, only pol III-transcribed R N A was able to form larger, 60-80S, R N P complexes which are thought to function in r R N A processing. M p p p A cap may therefore be required for proper assembly and/or function of the U3 snoRNP in plants (Kiss et aL, 1991). Therefore, it is likely that the T M G cap and the M p p p G cap, at least in the case of plant U3 snoRNA, are not functionally interchangeable. More recently, it has been shown that trans-splicing in trypanosomes requires methylation of the 5' end of the spliced leader R N A (Ullu and Tschudi, 1991), however whether this inactivation of trans-splicing is due to unmethylated 5' cap structure or lack of 2'-O-methylation is not clear.

7. F U T U R E PROSPECTS In vivo methylations of both D N A and R N A are very widespread in the biological systems. The effects of D N A methylations on gene expression are well documented in prokaryotes as well as eukaryotes (Razin and Cedar, 1991). Only recently the biological significance of the R N A methylations has begun to be realized. Very recently, increased expression of D N A methyltransferase has been shown to precede development of colonic neoplasia and represent an early event in cell transformation (EI-Deiry et al., 1991). Therefore, these methyltransferases will be potential targets for anti-cancer drugs. Studies on methylation of R N A and D N A will provide insights into regulatory aspects of cell metabolism. Acknowledgements--The studies from our lab are supported by grants from the U.S. Dept. of Health and

Human Services (GM38320). R. Singh is supported by the Leukemia Society of America fellowship. We thank Dr Shashi Gupta for help in preparing this review.

REFERENCES

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Methylated cap structures in eukaryotic RNAs: structure, synthesis and functions.

There are more than twenty capped small nuclear RNAs characterized in eukaryotic cells. All the capped RNAs appear to be involved in the processing of...
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