Cell, Vol. 63, 109-116,

October

5, 1990, Copyright

0 1990 by Cell Press

Monomethylated Cap Structures RNA Export from the Nucleus J&g Hamm and lain W. Mattaj European Molecular Biology Laboratory Meyerhofstrasse 1 D-6900 Heidelberg Federal Republic of Germany

Summary RNA export from the nucleus has been analyzed in Xenopus oocytes. Ul snRNAs made by RNA poiymerase II were exported into the cytoplasm, while Ul snRNAs synthesized by RNA poiymerase ill, and themfore with a different cap structure, remained in the nucleus. Export of the poiymerase Ii-transcribed RNAs was inhibited by the cap analog m7GpppG. Spliced mRNAs cawing monomethylguanosine cap structures were rapidly exported, while hypermethyiated cap structures delayed mRNA export. The export of a mutant precursor mRNA unable to form detectable spiicing complexes was also significantly delayed by incorporation of a hypermethyiated cap structure. The results suggest that the m7GpppN cap structure is likely to be a signal for RNA export from the nucleus. introduction in eukaryotic cells RNAs can be divided roughly into three groups, depending on which of the three RNA polymerases (pol I, pol II, pol ill) is involved in their transcription (Chambon, 1975; Roeder, 1976; Sentenac, 1965). Messenger RNA precursors (pre-mRNAs) and most of the nuclear U snRNAs (such as Ul-U5 snRNA or U7 snRNA) involved in pre-mRNA processing are transcribed by poi Ii. RNAs transcribed by pol Ii obtain a monomethylated, inverted guanosine cap structure (m7G(5’)ppp(5’)N) cotranscriptionaily. introns are removed from pre-mRNAs by the splicing machinery and the mature mRNAs are exported to the cytoplasm, while pre-mRNAs are usually found only in the nucleus (Steitz et al., 1966). The pol ii U snRNAs are transcribed as precursors that appear transiently in the cytoplasm, where the U snRNP proteins are made and stored (Eliceiri, 1974; Zeller et al., 1963). The pol II U snRNAs form U snRNPs in the cytoplasm and their m7GpppN cap structures are converted into m2m2s7GpppN cap structures (Mattaj, 1966; Reddy and Busch, 1966). The migration of the Ul and U2 snRNPs to the nucleus, their place of action, requires both the presence of the binding site for the common U snRNP proteins (Mattaj and DeRobertis, 1965) and the hypermethyiated cap structure (Fischer and Luhrmann, 1990; Hamm et al., 1990). Poi Ill RNAs show a more diverse intracellular distribution. Transfer RNAs are rapidly exported (De Robertis et al., 1962) after a defined and complex series of processing and modification steps (Melton et al., 1960). Experiments performed in Xenopus oocytes suggest that this transport might be carrier mediated (Zasioff, 1963). 5s RNA is also

Facilitate

exported into the cytoplasm of oocytes in a process that is separated from the assembly of preribosomal particles. This migration seems to require interaction of 55 RNA with TFIIIA or other proteins (Guddat et al., 1990). Other pol ill RNA% such as U6 and 7SK RNA, which differ in several respects from tRNA and 5S RNA (Murphy et al., 1969; Parry et al., 1969) are found exclusively in the nucleus. Although U6 snRNA is able to migrate into the nucleus when injected into the cytoplasm of oocytes (Hamm and Mattaj, 1969), it does not leave the nucleus following transcription from an injected U6 gene (Vankan et al., 1990). The potential of U6 to migrate into the nucleus, although not required in oocytes, might be essential in dividing ceils where nuclear components have to reaccumulate after mitosis. The factors influencing pol ii RNA export into the cytoplasm are poorly understood. it has been suggested that splicing complexes form during the transcription of pre-mRNAs (Beyer and Osheim, 1966). Studies of mRNA export in yeast (Legrain and Rosbash, 1969) and in mammalian cells (Chang and Sharp, 1969) have shown that mutations in pre-mRNAs that reduced their affinity for splicing factors result in increased export of unspliced pre-mRNAs. This implies that formation of splicing complexes might prevent pre-mRNA export. However, it was not clear whether the export of spliced mRNA or of pot ii pre-U snRNAs is a passive process, or whether it requires the recognition of signals present on these RNAs. In this work results suggesting that an m7GpppN cap structure might be an export signal for RNA are described. Results Design of a Poi iii Ul snRNA Gene When mutant or wild-type U6 snRNAs were transcribed from injected U6 genes in oocytes, none of the RNAs was found in the cytoplasm (Vankan et al., 1990). This implied that U6 snRNA, unlike Ul-U5 snRNAs, is not exported into the cytoplasm after transcription. One possible expianation for the differences in the intracellular migration of U6 and Ul-U5 snRNAs is that U6 snRNA is transcribed by poi Iii, while Ul-U5 snRNAs are transcribed by pol ii. if this were true, it might be possible to prevent the normal export of an RNA transcribed by poi Ii simply by converting it into a poi ill RNA. To test this hypothesis a poi iii Ul snRNA gene was designed to enable the synthesis of an RNA with the sequence of Ul snRNA, but made by pol Ill instead of by poi ii. As a consequence, the poi ii and poi iii Ul snRNAs should differ in the cap structure incorporated during transcription. Poi Ii RNAs obtain a monomethyiated, inverted guanosine cap structure (m’GpppN), while pol Iii RNAs retain a triphosphate Blend (pppG). The 5’fianking region of a Xenopus Ul snRNA gene was replaced by the 5’ flanking region of a human U6 gene (see Experimental Procedures for details). A G residue was inserted at position +l of the poi Ill Ul gene because it is the preferred

Cdl 110

4 POL

111 - 01

GENE:

r,

1

hU6-PRWOTER

lG+,l

Figure Genes

/ Ul snRNA

~TTTT

1

1. Construction

of Pol III Ul

The pol II promoter of a gene (Zeller et al., 1984) pol Ill promoter of a human U6 snRNA gene (Kunkel et al., 1986) (hlJ6 promoter). One G residue was inserted at the start site to provide the preferential start nucleotide of pol Ill. It was not necessary to introduce a termination signal for pol Ill because used has 4 T residues exactly at the end of the coding region.

start nucleotide of pol III. The generation of a pol Ill termination signal was not required because the Ul gene used already had a run of 4 T residues exactly at the beginning of the 3’ noncoding region (Figure 1). intracellular Location of Pol ii and Pol iii Ul snRNAs Pol II Ul snRNAs are exported into the cytoplasm and form U snRNPs before remigrating into the nucleus. This remigration requires the presence of a functional Sm binding site-the region required for interaction with the common U snRNP proteins-but not the binding sites of the Ul-specific proteins (Hamm et al., 1990). The distribution of Ul snRNAs between nucleus and cytoplasm was analyzed after injection of pol II or pol III Ul genes into nuclei of oocytes together with [aJ*P]GTP and a Xenopus 5S RNA gene as an internal control for transcription and RNA recovery. The pol Ill Ul genes were injected together with a-amanitin (1 ug/ml) to prevent any possible poi II transcription from the U6 promoter. Oocytes were dissected 12 hr later, and RNA was extracted from nuclear and cytoplasmic fractions or from total oocytes. A series of pol II Ul snRNAs, whose structures and protein binding properties are summarized in Table 1, was analyzed. In Ul AD, 6 nucleotides of the Sm binding site were substituted by an artificial sequence, but the binding sites for the Ul-specific proteins were present. U1.6ss carried the sequence required for nuclear migration of U6 snRNA instead of the Sm binding site, and in UlA361.6~~ the mutation 1.6s~ was combined with mutations destroying the binding sites for the Ul-specific proteins. The pol II Ulwt was present mainly in the nucleus (Figure 2A, bottom panel, pll Ulwt; the two RNAs migrating just above Ul snRNA are endogenous 5.6s RNA; see no Ul panel). The fraction found in the cytoplasm corresponded to pol II Ulwt forming Ul snRNPs before remigrating into the nucleus, since Ul snRNAs unable to

Table

1. Properties

of the Ul snRNA

RNA

Protein

wt

Ul-specific Sm Ul-specific Ulapecific -

+

-

Ul-specific Sm Ul-specific

+

S124-129AJAAUUU-CUCGAG S126-130lAUUUCUACUA S27, 29, 30/U, A, U-G, C, C S66-70/GCA-UAU S126-13O/AUUUWJACUA Al-11

AD U1.6ss UlA3Bl.Gss

UlwtA.5’ Ul ADA5’

Binding

Mutants

Mutation

Al-11 S124-129AJAAUUU-CUCGAG

snRNA

Xenopus Ul snRNA was replaced by the of transcription (G+,) the Ui snRNA gene

bind either the Sm proteins (Ul AD, Ul.Gss) or both the Sm proteins and the Ul-specific proteins (UlA3816ss) accumulated in the cytoplasm, being unable to remigrate into the nucleus (Figure 2A, bottom panel). In contrast, all pol III Ul snRNAs were found exclusively in the nucleus, independent of the presence of an Sm binding site (Figure 2A, plil Ul, upper panel). The pol Ill Ulwt RNA consistently accumulated to a lower level than the mutant pol Ill Ul snRNAs (see below). The distribution of the pol Ill Ul snRNAs implied that they were not exported into the cytoplasm. However, it was possible that the cytoplasmic half-life of pol Ill Ul snRNAs was too short to result in their detection in the cytoplasm. In that case the pol Ill Ulwt RNA should be precipitable by anti-Sm antibodies, because the Sm proteins would have bound to the RNA during its transient passage through the cytoplasm. To test this possibility, oocytes were injected with pol II Ulwt or pol Ill Ulwt or mutant genes together with [a-s*P]mP and 55 genes. Oocytes were dissected 12 hr later, and the nuclear fractions were used for immunoprecipitation with anti8m and anti-trimethylguanosine cap antibodies (anti-TMG). Nuclear fractions were used to exclude an artifactuai association of Sm proteins with Ulwt RNA after mixture of nuclear and cytoplasmic components. The pol II Ulwt was precipitated by anti-Sm and anti-TMG cap antibodies, while none of the pol III Ul snRNAs was immunoprecipitated (Figure 28, IPP). This observation allowed the conclusion that the pal III Ulwt had not been transiently in the cytoplasm. However, the possibility remained that pol Ill Ul snRNAs left the nucleus slowly but were undetectable, owing to cytoplasmic instability. We noted that pol Ill Ul snRNAs, like in vitro-synthesized uncapped RNAs normally made by pal II, were less stable than capped pal II Ul snRNAs when injected into the cytoplasm (Hamm et al., 1990; data not shown). “Natural” pol III RNAs protect their 5’ and 3’ ends by the formation of base-paired stem structures. The single-stranded S’end of Ul RNA (nucleotides l-11) was therefore deleted to enhance the stability of the pol Ill Ul snRNAs. This deletion (UlA5’) was introduced into pol II and pol Ill Ul snRNA genes to allow direct comparison of the migration properties of the corresponding RNAs. Pol II UlwtA8, pol II UlADA5’, or pol Ill UlwtA5’ genes were injected into nuclei together with [a-sP]GTP and 5s RNA genes, and the intracellular location of the RNAs was determined 12 hr later. Again, the pol Ill UlwtA5’ RNA was found exclusively in the nucleus, while the pol II UlA5’ RNAs were exported into the cytoplasm (Figure 3A). In fact, the pol II UlwtA5’ RNA accumulated in the cytoplasm to an even greater extent than the pol II Ulwt RNA, although it had

m’GpppN 111

Cap Structure

As RNA

Export

Signal

A Lf1lVt T

C

U1.6SS

UlAD N

T

C

N

T

C

UlA3B1.6SS N

T

C

no Ul N

T

C

N

5.8s

Plll Ul

5.0s

pll Ul

5s

Figure antl-Sm

antl-TMG

II

Ul SN

5s

aI&

LJ1

IPP

both a trimethylated cap and bound Sm proteins (data not shown). This might have been due to a decrease in the accessibility of the TMG cap structure, which has been shown to be required to signal nuclear migration (Fischer and Luhrmann, 1990; Hamm et al., 1990). The pol Ill UlwtA5’ RNA also provided a possible explanation for why the amount of pol III Ulwt was reproducibly lower than that of the other pol Ill Ul snRNAs. Fol Ill apparently terminated in the U-rich Sm binding site generating the truncated product Ul’ (Figure 3A), since this shorter product was not observed with pol Ill UlADA5’where the Sm bind-

2. Characterization

of Ul snRNAs

Transcribed

by RNA Pol Ill

(A) intracellular location of pol II and pol Ill Ul snRNAs. A series of wildtype and mutant VI snRNA genes (the structures and protein binding properties of the corresponding Ul snRNAs are described in Table 1) carrying either a pol II (pll Ul) or a pol Ill (plll Ul) promoter were injected into the nucleus of oocytes together with [w~P]GTP and a Xenopus 55 RNA gene (as an internal control for transcription efficiency and RNA recovery). For the injections of the pol Ill Ul snRNA genes a-amanitin (1 ug/ml) was included. A mixture containing a-amanitin, 55 DNA, and [a-32P]GTP, but no Ul DNA, was injected to show endogenous 5.8s RNA (no Ul). The absolute amount of 5.8s RNA made is variable. The cytoplasmic RNA migrating slightly slower than the Ul snRNA present in the fractions of pill Ul AD and pill UlA8Bl.6ss is predominantly 5.8s RNA. Oocytes were dissected manually 12 hr after injection, and RNA was extracted from nuclear (N) and cytoplasmic (C) fractions or from total oocytes (T). One oocyte equivalent of RNA was separated on denaturing acrylamide gels (This figure is the result of a single experiment. Gaps were introduced afterward to facilitate the identification of individual RNAs.) (B) lmmunoprecipitation of pol II and pol Ill Ul snRNAs from nuclear fractions. Oocytes were injected with a pol II Ulwt gene or a pol Ill wildtype or mutant gene together with a-amanitin (only with pol Ill genes), 55 DNA, and [a-=P]GTl? Ten oocytes were dissected 12 hr later, and Ul snRNPs were immunoprecipitated from nuclear fractions with antiSm or anti-TMG cap antibodies. RNA present in the supernatant at the end of the immunoprecipitation (SN) and in the immunoprecipitate (IPP) were extracted and analyzed on a denaturing acrylamide gel.

ing site was substituted by a GC-rich sequence (data not shown). In the presence of the 5’ end, this product is presumably produced from pol Ill Ulwt, but is unstable. The pol Ill UlwtA8 RNA was purified from acrylamide gels and reinjected into the cytoplasm of oocytes. The RNA was coinjected with Ul AD and U5 snRNA synthesized by T7 RNA polymerase in the presence of cap analogs (m’GpppG). These T7 RNAs served as internal controls for RNA stability and for nuclear migration. Ul AD is unable to enter the nucleus (no Sm binding site), while U5 migrates into the nucleus. Pol Ill UlwtA5’ was stable for

Cell 112

8

pill

wtA5’

I To

I T

C

N

UlAD

UlA5’

NC Figure

u5

Figure 3. Characterization of Ul Stranded 5’ End of the RNA (UlA5’)

snRNAs

Lacking

the

Single-

(A) Intracellular location of UlA5’ RNA. The sequence corresponding to the single-stranded 5’ end of Ul snRNA (nucleotides l-11) was deleted in the pll wt. pll AD, and pill wt genes. These genes were injected into oocytes together with a-amanitin (only with pill wt), 55 DNA, and [a-*P]GTP Oocytes were dissected manually 12 hr after injection, and RNA was extracted from nuclear(N) and cytoplasmic (C) fractions or from total oocytes (T). One oocyte equivalent of RNA was separated on a denaturing acrylamide gel. A shorter transcript (Ul’) from the pill UlwtAB’gene is probably due to termination of pol Ill in the Sm binding site. (B) Cytoplasmicstabilityof pol Ill Ul wtA5’RNA. Oocytes were injected with a pill UlwtAB gene together with a-amanitin and [a-32P]GTP RNA was extracted 12 hr later from 12 oocytes, separated on a denaturing acrylamide gel, and the pill Ulw!AS RNA was eluted from the gel. The purified pol Ill UlwtAS RNA was injected into the cytoplasm of oocytes together with Ul AD and U5 snRNA made by T7 RNA polymerase (including a monomethylated cap analog). Oocytes were dissected manually 12 hr after injection, and RNA was extracted from nuclear(N) and cytoplasmic (C) fractions or from total oocytes either directly after the injection (Ts) or after 12 hr (T).

more than 12 hr in the cytoplasm of oocytes (Figure 38). This demonstrated that the difference in intracellular location observed for pol II and pol Ill Ul snRNAs was not due to different cytoplasmic stabilities of the RNAs. These experiments provided strong evidence that pol III Ul snRNAs do not leave the nucleus, while pol II Ul snRNAs are exported into the cytoplasm. Compared with completely processed pol II Ulwt RNA, the pol III Ul snRNAs will carry several U residues at their 3’ ends. However, pol II Ul snRNA precursors also have additional nucleotides at their 3’ ends, which are removed after export from the nucleus. In the case of the pol II Ul snRNAs studied here, this extension includes a run of 4 U residues.

4. Inhibition

of Pal II Ul Export

NC by Cap Dinucleotide

Oocytes were injected with a pol II UIAD gene. Sixteen hours later [a-=P]GTP was injected together with either HsO or 50 mM m7GpppG. After 6 hr further incubation nuclear and cytoplasmic fractions (denoted N and C below the figure) were separated, and RNA was analyzed on a denaturing acrylamide gel. Lane 1, control injection, nuclear fraction; lane 2, control injection, cytoplasmic fraction; lane 3, 50 mM m7GpppG injection, nuclear fraction; lane 4, 50 mM m7GpppG injection, cytoplasmic fraction. The large ribosomal RNAs (poorly resolved in this gel system), the ES precursor to 5.5s RNA, and Ul AD transcripts are indicated.

Inhibition of Pal II Ul Export by Cap Analog The difference in export of the pol II and pol Ill Ul snRNAs could therefore be due either to their different cap structures or to the RNA polymerase involved in their transcription. A direct test of cap involvement was carried out, analogous to an experiment used to analyze the role of the TMG cap in Ul snRNA transport from the cytoplasm into the nucleus (Fischer and Liihrmann, 1990). Genes encoding pol II Ul AD, chosen because it remains in the cytoplasm after export (see Figure 2A), were injected into the nuclei of oocytes. Sixteen hours later [a-32P]GTP was injected either alone or in the presence of dinucleotide cap analog (m’GpppG). After a further 6 hr oocytes were dissected, and RNA from nuclear and cytoplasmic fractions was analyzed. In control oocytes (Figure 4, lanes 1 and 2) pol II Ul AD transcripts were present almost exclusively in the cytoplasmic fraction (lane 2) as previously observed when oocytes were fractionated after 12 hr (see Figure 2A). In contrast, in oocytes injected with 50 mM mrGpppG, Ul AD transcripts were largely nuclear (Figure 4, lane 3). The cap dinucleotide did not affect the intracellular location of the large ribosomal RNA-s, which are pol I transcripts and have triphosphate 5’ ends, or of the 8S precursor to 5.8s RNA (Figure 4, lanes l-4). This inhibitory effect is both specific and saturable since neither m2~*~7GpppG at 50 mM nor mrGpppG at 5 mM inhibit UlAD export from the nucleus (data not shown).

r$:pppN

Cap Structure

As RNA Export

Signal

b cu

Figure 5. intracellular Location Capped Splicing Products

i 0 2 1 m/90’ -TC

1 m/l 50’ NT

C

2m190’ N

3m/90’

II TCNT

CN

P b

EIZ

Splicing of Capped Pn+mRNAs To uncouple the effect of cap structure on RNA transport entirely from effects related to the transcription of the RNA, export of synthetic spliced mRNAs carrying preformed cap structures was analyzed. The effect of incorporating mono- (lm), di- (2m), or trimethyiated (3m) cap structures into a model pre-mRNA (pBSAd1, Konarska and Sharp, 1987) on the export of the spliced mRNA product was tested. The analysis showed that the spliced lm mRNA left the nucleus rapidly (Figure 5, lm/90’, lm1150’) after completion of the splicing reaction, which proceeds for 80-70 min after injection of pre-mRNA (Hamm et al., 1989). The pre-mRNA present in the cytoplasm was likely to be due to unspecific leakage just after injection into the nucleus, because the amount of cytopiasmic pre-mRNA was highly variable between individual experiments. This leakage can apparently only occur for a short time after injection, because the intron lariat, produced later in the splicing reaction, is never seen to leave the nucleus. When 2mAdl or 3mAdl pre-mRNAs were injected into nuclei, both were spliced efficiently. No differences in the rate or the extent of the splicing reaction were observed in repeated experiments (Figure 5; data not shown). in addition, they did not appear to lose their cap structures, because the spliced 3mAdl mRNA could be precipitated by anti-TMG cap antibodies (Figure 5, a-TMG). However, the export of spliced PmAdl and 3mAdl mRNA was delayed. After 90 min 3mAdl mRNA was present only in the nucleus, while 30%1 of the 2mAdl mRNA and 80%-80% of lmAd1 mRNA was found in the cytoplasm (Figure 5, E,*). Even after 150 min 3mAdl mRNA was still mainly in the nucleus (data not shown). In ail experiments the lariat intron (I) was nuclear, providing a control for the integrity of the nuclear membrane. The tested cap structures did not affect the splicing reaction directly. Rather, the substitution of the normal m7GpppN cap structure by hypermethyiated cap structures interfered with the export of the spliced mRNA,

of Differently

Ad1 pre-mRNAs were synthesized in the presence of mono-(lm), di- (2m), or trimethylated (3m) cap analogs. Each of the premRNAs was injected into the nucleus of oocytes, and oocytes were dissected 90 min or 150 min later. RNA was extracted from nuclear (N) and cyto plasmic (C) fractions or from total oocytes (T). One oocyte equivalent of RNA was separated on a denaturing acrylamide gel (p, pre-mRNA; I, intron lariat; E12, spliced mRNA). To determine whether the TMG cap was maintained during splicing, total RNA was extracted and purified from 10 oocytes 20 min after injection of 3mAdl pm-mRNA. RNA was immunoprecipitated with anti-TMG cap antibodies from the purified RNA (a-TMG T/3m/20’).

showing that the effect of an mrGpppN cap on RNA export could be uncoupled from transcription. Export and Splicing Complex Formation of Mutated Pre-mRNAs Experiments performed in yeast (Legrain and Rosbash, 1989) and in mammalian ceils (Chang and Sharp, 1989) indicated that the formation of splicing complexes might prevent the export of pre-mRNA. It was therefore necessary to show that the effect of changing the pre-mRNA cap structure on export was not due to an effect on spiiceosome disassembly after completion of the splicing reaction. A series of mutated Ad1 pre-mRNAs was constructed for this experiment (Figure 8A). Ads+s carried a point mutation near the 5’ splice site, and AdA5' lacked most of the Ad1 sequence, including the 5’splice site, but retained the branch point, the poiypyrimidine tract, and the 3’ splice site. AdA3' was a truncated form of Ad1 that had lost the branch point, the polypyrimidine tract, and the 3’ splice site, but retained the 5’ splice site. Because the mutated pre-mRNAs were of different length, they could be coinjetted into nuclei and analyzed simultaneously. The rationale of this experiment was that a mutated pre-mRNA unable to form the first splicing intermediate, complex A, should leave the nucleus, while pre-mRNAs blocked in subsequent steps of spliceosome assembly could be specifically retained because they might form dead-end complexes. The cap structure of a pre-mRNA unable to form splicing complexes could then be changed, which would allow separation of effects related to spliceosome assembly or disassembly from RNA export. The mutated Ad1 pre-mRNAs were synthesized in vitro. All RNAs carried monomethyiated cap structures; AdA3' was also made with a trimethylated cap structure. Splicing complexes formed on the pre-mRNAs in oocytes were examined. Oocytes were homogenized 10 min after premRNA injection, and splicing complexes were analyzed on native gels. Adlwt pre-mRNA was also injected to ob-

GloA L

I

6

5’55

bp

I

I

Ad S+5

3’5s

AdA3’

235 1

I

1

bp

3’5s

Ad85

I

C EEEEE 7 --m-7

lm -T

AdA3’ C

3m AdA3’ N

T

C

N

lm Ads+5

lm

B Figure

e

AdA3’

3m

AdA3’

lm

AdA5’

D 6. Export

and Splicing

Complex

Formation

of Mutant

Pre-mRNA

(A) Structures of wild-type and mutant pm-mRNAs. The positions of the 5’ splice site (~‘ss), the branch point (bp), and the 3’ splice site (39s) in Ad s+s carries a G to A mutation in the 5’-splice site at intron position the Adlwt pre-mRNA are indicated (1, start nucleotide; 365, 3’end nucleotide). +.5. AdA3’ is a truncated form of Adl, lacking the branch point and the 3’ splice site. AdA5’ is lacking the 5’ end of Adl, induding the 5’ splice site, but still carries the branch point and the 3’ splice site. 1mAd s+s, ImAdAS’, (8) Splicing complex formation. ImAdlwt, ImAdAS’, or 3mAdA3’ was injected into nuclei of oocytes. Five oocytes were homogenized 10 min later in J buffer, and one oocyte equivalent was analyzed on a native gel. Splicing complexes formed by the Adlwt pre-mRNA are labeled A, B, and C. (C) Total RNA was extracted from the remaining homogenate and analyzed on a denaturing gel. (D) Intracellular location of mutant pm-mRNAs. Mixtures of Ad s+s, AdA3’, and AdA5’ were injected into nuclei of oocytes. Ads+s and AdA5’ always had a monomethylated cap structure (lm), while AdA3’ had either a mono- (lm) or a trimethylated (3m) cap structure. Oocytes were dissected 2 hr after injection, and RNA was extracted from nuclear (N) and cytoplasmic (C) fractions or from total oocytes (T). One oocyte equivalent of RNA was separated on a denaturing acrylamide gel. A fraction of the Ad s+s was spliced using an unidentified cryptic splice site, generating the products labeled E12* (mRNA) and I* (intron). This cryptic splice site is used to an extremely variabte extent, depending on the oocyte batch used (observed efficiencies, 0%-20%).

~~~pppN

Cap Structure

As RNA Export

Signal

RNA

slow export (2mml Ad, nlRNA)

Figure 7. Model for the Order

of Events

Preceding

RNA Export

RNA features influencing the export route used are boxed. The presence or absence of a particular feature is indicated schematically (-, absence; +, presence). Examples of RNAs using a certain route are given in parentheses at the end of each route. An asterisk indicates a mutated form of an RNA. 53s’ represents a pre-mRNA with a mutated 5’ splice site, but it can also represent a splice site regulated by a trans-acting factor. The unidentified factor facilitating tRNA export is represented by X.

tain markers for the position of the normal splicing complexes (A, 6, and C; Hamm et al., 1989). Ads+s and AdA5’ formed complexes comigrating with the A complex and small amounts of complexes comigrating with the B complex. It has previously been demonstrated that the 5’ splice site is not required for early steps of spliceosome assembly in vitro (Frendewey and Keller, 1985; Lamond et al., 1987). The AdA3’ pre-mRNAs did not form detectable complexes independent of the cap structure incorporated (Figure 6B). To verify that similar amounts of pre-mRNAs had been injected and that they were stable during the course of the experiment, total RNA was extracted from the remaining homogenate and analyzed on a denaturing gel (Figure SC). AdA3’, carrying either a monomethylated or a trimethylated cap structure, was mixed with Ads+s and AdAY, which both carried a monomethylated cap structure. These mixtures were injected into the nuclei of oocytes, and the intracellular location of RNAs was determined 2 hr later. Ads+s and AdA5’, the pre-mRNAs forming splicing complex A, were retained in the nucleus, as expected. Surprisingly, Ads+5 was spliced .inefficiently using an unidentified cryptic splice site, generating the splicing products I’ (lariat intron) and E12* (spliced mRNA). These splicing products showed the normal distribution: the intron was in the nucleus, while the mRNA was exported into the cytoplasm (Figure 8D). AdA3’, the pre-mRNA unable to form detectable splicing complexes, was exported to a significant extent when carrying a monomethylated cap structure (Figure 8D, lmAdA3’). The amount of AdA3’ex-

ported was greatly reduced by incorporating a trimethylated cap structure (Figure SD, 3mAdA3’). Thus, in the absence of possible interference by either transcription or splicing complex formation, a strong effect of changing the m7GpppN cap structure to m2s2s7GpppN on RNA export could be detected. Discussion The results obtained in this study, together with previous work, suggest that there are several factors that influence RNA export from the nucleus. These are summarized in Figure 7. Two major factors are the cap structures of the RNAs and whether they contain introns. Because the cap structure of an RNA depends primarily on the RNA polymerase by which it is transcribed, triphosphate-capped pol III RNAs and pol II RNAs with m7GpppN cap structures will be discussed separately. Pal III RNAs Ul snRNAs of identical sequence but synthesized by either pol II or pol III showed different intracellular distributions. Pol II Ul snRNAs were exported to the cytoplasm, while pol III Ul snRNAs remained in the nucleus. U6 snRNA, which is transcribed by pol III, is also not exported when transcribed from injected U6 genes in oocytes (Vankan et al., 1990). In contrast, other pol Ill RNAs, such as 5S RNA and tRNA, are located in the cytoplasm. The transport of 55 RNA seems to be dependent on its ability to bind to certain proteins, since mutated 5s RNAs unable

Cdl 116

to interact with these proteins remained in the nucleus (Guddat et al., 1990). Similarly, earlier work had shown that the export of tRNA can be severely affected by single point mutations, including at least one that was unlikely to change the secondary or tertiary structure of the tRNA (mutation U57), indicating that tRNA requires interaction with an unidentified factor in order to be exported (Zasloff, 1983). Taking into account the results obtained with 5S RNA, this unknown factor might be a tRNA binding protein. All the results discussed in this section would be compatible with a model in which pol Ill RNAs are not exported into the cytoplasm unless they interact with specific proteins to form an “export RNP” (Figure 7). Pal II RNAs Pol II RNAs can be divided into two classes. Group I contains RNAs without introns or with mutated introns that do not form splicing complexes. Group II contains RNAs with introns or with mutated introns that can form splicing complexes but cannot be spliced. Group I RNAs are rapidly exported. This group contains the precursors of Ul-U5 snRNAs (pre-Ul-U5) and pre-mRNAs lacking the branch point/polypyrimidine tract region. Rapid export seems to depend upon the m7GpppN cap structure. The importance of the cap structure was most dramatically demonstrated by comparison of pol II and pol Ill Ul snRNAs. The pol II Ul snRNAs carried a m7GpppN cap structure and were efficiently exported. The pol Ill Ul snRNAs had a pppG 5’end and remained in the nucleus. The possibility that pol Ill RNAs are trapped in a nuclear subcompartment used for pol Ill transcription seems unlikely since 5s RNA and tRNA readily leave the nucleus when transcribed in oocytes. Also, export of wild-type tRNA and the selective nuclear retention of tRNA carrying a specific point mutation (U57) are maintained when the purified, processed tRNAs are injected into the nucleus of oocytes (Zasloff, 1983). Both the inhibition of export of pol II Ul RNAs by cap dinucleotides and the decreased rate of export of mRNAs with hypermethylated cap structures support models where m7GpppN cap structures are involved in RNA export. Spliced mRNAs with hypermethylated cap structures were exported slowly, and mutant pre-mRNAs unable to form splicing complexes were only exported rapidly when they carried an m7GpppN cap structure. Order of Events Regulating mRNA Export Pre-mRNA and mRNA are found in different intracellular compartments: spliced mRNA is exported, while premRNA remains in the nucleus. Similarly, for polyadenylated mRNAs, cleavage and polyadenylation seem to be prerequisites for RNA export (Wickens and Gurdon, 1983). The fact that splicing complex formation and pre-mRNA export are opposing processes was demonstrated by elegant experiments performed with yeast. It was shown that unspliced pre-mRNA was only exported if early steps of splicing complex formation were disturbed. This could be achieved either by mutating highly conserved splicing signals or by mutating &Ins-acting factors involved early in the formation of splicing complexes. Interference with

later steps in complex formation resulted in the retention of pre-mRNA in the nucleus, probably owing to the formation of dead-end complexes (Legrain and Rosbash, 1989). Similar conclusions were reached by Chang and Sharp (1989), who studied the effect of intron mutations on premRNA export in mammalian cultured cells. However, for technical reasons, it was not possible to demonstrate directly the formation of “blocked” splicing complexes that interfered with export of mutated pre-mRNAs. Rather, a correlation was made between the abilities of pre-mRNAs to form splicing complexes in vitro and the export of unspliced pre-mRNAs in vivo. In Xenopus oocytes both processes could be analyzed in the same cell. Mutated pre-mRNAs that could not be spliced but that could form splicing complexes were trapped in the nucleus, presumably in dead-end complexes. Conversely, a pre-mRNA unable to form detectable splicing complexes was exported rapidly into the cytoplasm. Comparison of the Roles of Pal II Cap Structures From the experiments described here and from studies of migration of Ul snRNP from the cytoplasm to the nucleus (Fischer and Liihrmann, 1990; Hamm et al., 1990), it apears that the primary functional difference between the monomethylguanosine and trimethylguanosine pol II RNA cap structures is to determine the rate and the direction of RNA transport. Although previous studies (Konarska et al., 1984; lnoue et al., 1989) on the influence of the cap structure on splicing indicated that cap structures might affect splicing efficiency, they did not show that an m7GpppN cap is an absolute requirement for splicing. The experiments described here with pre-mRNAs having mono-, di-, or trimethylated cap structures indicate that splicing is not affected by the presence of a hypermethylated cap structure. There is no doubt that mRNA cap structures have a function in the translation of mRNA (Shatkin, 1978; Kozak, 1989). Nevertheless, there are several examples of mRNAs that are translated but do not carry an m7GpppN cap structure. Of particular interest here is the fact that the presence of hypermethylated cap structures does not prevent translation. Certain viruses produce mRNAs with dimethylguanosine or trimethylguanosine cap structures that are translated in host cells (Hsu-Chen et al., 1978; van Duijn et al., 1988). Hypermethylated cap structures also allow the translation of mRNAs in vitro, although they alter the affinity for cap binding proteins and translation efficiency (Darzynkiewicz et al., 1988). In addition, it has been shown that Caenorhabditis elegans mRNAs produced by frans-splicing retain a TMG cap structure but are nevertheless associated with polysomes (Liou and Blumenthal, 1990; Van Doren and Hirsh, 1990). It would be interesting to measure the rate of export from the nucleus of these RNAs. Based on this information it seems reasonable to suggest that the major differential function of monomethylated and trimethylated pol II caps is to influence the rate and the direction of transport through the nuclear membrane. The mechanism by which this occurs is currently unknown. However, the fact that the injection of m7GpppG

m7GpppN 117

Cap Structure

As RNA Export

Signal

dinucleotide inhibits export of pol H-transcribed Ul snRNA (Figure 4) and that the TMG cap analog can inhibit nuclear import of U snRNAs (Fischer and Lijhrmann, 1990) indicates that cap recognition by a rrans-acting factor is involved. Nuclear, as well as cytoplasmic, cap binding proteins have been described (Patzelt et al., 1983; Shatkin, 1985). There are many possible ways in which cap binding proteins might be involved in RNA export. For example, they might act as receptors on the inside of the nuclear pore to recognize capped RNAs prior to export or as soluble adaptors that could bind both to the cap and to elements of the transport machinery. Alternatively, the proteins could shuttle between nucleus and cytoplasm carrying bound RNA with them, playing a role analogous to that of TFIIIA in the export of 5s RNA from the oocyte nucleus described above. Further experiments to explore these possibilities are in progress.

using IPPra (Hamm et al., 1967). Splicing complexes were analyzed on native gels as described (Hamm et al., 1969). Cap analog (m7Gpp pG, sodium salt, PL Biochemicals) was dissolved in HsO and injected into oocytes at a final concentration of 50 mM or 5 mM.

Antlbodles A monoclonal anti-Sm antibody (Y12, Lerner et al., 1961) and a polyclonal anti-TMG cap antiserum (Liihrmann et al., 1962) were used.

Acknowledgments We wish to thank E. Darzynkiewicz and S. Tahara for modified cap analogs, R. Luhrmann for antiTMG cap antibodies, Gary Kunkel for the human U6 gene, Huw Parry for generating the hU6 EcoRV gene, Helen Frey for secretarial help, and Angus Lamond, Jordi Bernues, Daniel Scherly, and Kenneth Simmen for comments on the manuscript. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 16 USC Section 1734 solely to indicate this fact. Received

Experimental

Procedures

Constructlon

of a Pol III Ul Gene

An EcoRV restriction site was introduced directly upstream of the start site of transcription of a human U6 snRNA gene (Kunkel et al., 1966). This was achieved by changing the sequence AACACC (nucleotides -6 to -1, relative to the coding region) to GATATC. The sequence GATATCG was inserted directly in front of the coding region of a Xenopus Ul snRNA gene (Zeller et al., 1964). Both mutations were introduced by site-directed mutagenesis (Kramer et al., 1964). The generation of the EcoRV restriction sites enabled the replacement of the sequence downstream of position +l of the hU6 snRNA gene by the sequence containing the coding and 3’ flanking region of the XUl snRNA gene. The genes coding for pol Ill UIAD, pol Ill Ul.Gss, pol Ill UlA3Bl.Gss, and pol Ill Ul A5’were generated by introducing the corresponding mutations (see Table 1) into the EcoRV Ul snRNA gene and subsequently replacing the B’flanking region by that of the hU6 snRNA gene.

Generation

of Mutant

Pre-mRNAs

The gene coding for Adscg was constructed by introducing a G to A change at position +5 of the intron 1 sequence of pBSAd1 (Konarska and Sharp, 1967) by site-directed mutagenesis. The Ads+5 gene was inserted into the vector GEM4. The RNA was synthesized with T7 RNA polymerase after cleavage of this DNA with the restriction enzyme SauM. The AdAS’gene was generated by deletion of a Hindlll-Hindlll fragment of pBSAd1. The templates for T3 RNA polymerase transcription of pBSAd1 and AdA5’were linearized by cleavage with the restriction enzyme SauSA. AdA3’ pre-mRNA was generated by cleavage of pBSAd1 DNA with the restriction enzyme Fnu4Hl and subsequent transcription with T3 RNA polymerase. T7 and T3 RNAs were synthesized as described previously (Hamm et al., 1967). The synthetic RNAs were diluted (1:lO) for oocyte injections.

Oocyte

Injectlons

To transcribe

RNAs 20 nl of a solution containing plasmids carrying the Ul snRNA gene (0.5 &d), a 5S RNA gene (X1&60764, Pieler et al., 1965) (1 nglul). [a-32P]GTP (5 uCi/ul), and, only for injections of pol Ill Ul snRNA genes, a-amanitin (1 ng/ul) was coinjetted into nuclei of oocytes. For nuclear injections of purified RNAs or T7 or T3 RNAs 10 nl was injected per nucleus. Oocytes were treated with collagenase, and nuclei were visualized before nuclear injections, as described previously (Kressmann and Birnstiel, 1960). To determine the intracellular location of RNAs, five to six oocytes were dissected manually in J buffer (70 mM NH&I. 7 mM MgCIs, 0.1 mM EDTA, 2.5 mM dithiothreitol, 20 mM Tris-HCI [pH 7.51, 10% glycerol). RNA was extracted by homogenizing total or dissected oocytes in homomedium (50 mM Tris-HCI [pH 7.51, 5 mM EDTA, 1.5% SDS, 300 mM NaCI, 1.5 mg/ml proteinase K), extracting proteins once with phenol and once with phenol-chloroform (l:l), and precipitating RNAs with 3 vol of ethanol. lmmunoprecipitations were performed as described previously

corresponding

May

14, 1990; revised

July 17, 1990.

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