nucleolar protein required for pre-rRNA processing and ... - Europe PMC

The EMBO Journal vol. 1 3 no. 1 3 pp.3136 - 3148, 1994

Synthetic lethality with fibrillarin identifies NOP77p, a nucleolar protein required for pre-rRNA processing and modification Thierry Berges, Elisabeth Petfalski, David Tollervey and Eduard C.Hurt1 EMBL, Meyerhofstrasse 1, Postfach 1022.09, D-69117 Heidelberg, Germany 'Corresponding author Communicated by K.Simons

The nucleolar protein fibrillarin (encoded by the NOPI gene in yeast), is required for many post-transcriptional steps in yeast ribosome synthesis. A screen for mutations showing synthetic lethality with a temperature sensitive nopl-S allele led to the identification of the NOP77 gene. NOP77 is essential for viability and encodes a nucleolar protein with a predicted molecular weight of 77 kDa. Depletion of NOP77p impairs both the processing and methylation of the pre-rRNA. The processing defect is greatest for the pathway leading to 25S rRNA synthesis, and is distinctly different from that observed for mutations in other nucleolar components. NOP77p contains three canonical RNA recognition motifs (RRMs), suggesting that it is an RNA binding protein. The NOP77 allele which complements the synthetic lethal nopi strains has an alanine at position 308, predicted to lie in helix al of RRM3, whereas the non-complementing nop77-1 allele contains a proline at the corresponding position. We propose that NOP77p mediates specific interactions between NOPlp and the pre-rRNA. Key words: fibrillarin/NOP1/nucleolus/RNA binding/yeast

Introduction In eukaryotes, ribosome biogenesis takes place in the nucleolus, a specialized nuclear compartment (Hadjiolov, 1985; Warner, 1990). Within the nucleolus, transcription of the rRNA genes produces an rRNA precursor (pre-rRNA) which is processed by modification and cleavage to give the mature rRNAs. During the transcription and processing of the pre-rRNA, ribosomal proteins assemble with the mature rRNA regions to form the large and small ribosomal subunits. The various steps in ribosome synthesis require resident nucleolar proteins as well as non-ribosomal RNAs (small nucleolar RNAs or snoRNAs) (for reviews see Tollervey and Hurt, 1990; Warner, 1990; Fournier and Maxwell, 1993; Mattaj et al., 1993). Although components involved in the various steps of ribosome biogenesis have been identified, little is known about their interactions or specific functions. The essential yeast nucleolar protein, NOPip, is highly homologous (70% identity) to the vertebrate nucleolar protein, fibrillarin (Lapeyre et al., 1990; Aris and Blobel, 1991; Jansen et al., 1991). Moreover, expression of fibrillarin from human or Xenopus can functionally replace NOPIp in a yeast

strain deleted for NOPI (Jansen et al., 1991), demonstrating functional conservation during evolution. NOPlp is associated with all known yeast snoRNAs (Schimmang et al., 1989), with the exception of the RNA component of RNase MRP (P.Mitchell and D.Tollervey, unpublished results). Similarly, fibrillarin is associated with several vertebrate snoRNAs (Lischwe et al., 1985; Tyc and Steitz, 1989). Genetic depletion of NOPip leads to a severe inhibition of pre-rRNA processing, affecting mainly 18S rRNA production, and also inhibits pre-rRNA methylation (Tollervey et al., 1991). Surprisingly, temperature-sensitive (ts) mutants of the NOPI gene have phenotypes which do not closely resemble the effects of depletion of NOPlp. Different ts alleles are specifically impaired in distinct steps in ribosome biogenesis, showing defects in methylation of the pre-rRNA, pre-rRNA processing and assembly of the ribosomal subunits (Tollervey et al., 1993). Mutants carrying nopl-2 or nopl-5 fall to synthesize any pre-rRNA species except the 35S primary transcript and are therefore inhibited in both 18S and 25S rRNA synthesis. In the case of nopl-5 mutants, this inhibition is greater for 25S than 18S rRNA. In contrast, the nopl-3 mutation results in the inhibition of the methylation of the pre-rRNA, but does not strongly inhibit processing. This shows that pre-rRNA methylation is not required for correct processing. However, since nopl-3 is a tight ts-lethal mutation, rRNA methylation is presumably required for the function of yeast cytoplasmic ribosomes. Methylation of the yeast mitochondrial large subunit rRNA is also essential for its function (Sirum-Connolly and Mason, 1993). Finally, the nopl-4 and nopl-7 mutations inhibit late steps in the assembly of the 60S ribosomal subunits. From this it is clear that NOPIp is required at many steps in ribosome biogenesis. We believe that NOPlp is a component of higher order complexes made up of the pre-rRNA, ribosomal proteins, small nucleolar ribonucleoprotein particles (snoRNPs) and other nucleolar proteins, which bring about the cleavage, modification and assembly reactions in ribosome synthesis. Consistent with this model, NOPlp can be detected in association with the essential U3-snoRNP protein, SOFlp (Jansen et al., 1993). Repeated attempts to isolate extragenic suppressors of the ts alleles of NOP] were unsuccessful, although intragenic suppressors could readily be obtained. To identify components which functionally interact with NOPlp we therefore screened for synthetic lethal (sl) mutants. Synthetic lethality provides in vivo evidence that two gene products physically interact with each other or function in overlapping pathways (Huffaker et al., 1987; Bender and Pringle, 1991; Wimmer et al., 1992, 1993). A collection of recessive mutants (lossof-function) were isolated which are lethal in combination with nopl-S, but viable with wild-type NOPI. Many of these strains are complemented by a cloned NOP77 allele encoding a novel nucleolar protein NOP77p, which is itself required for normal pre-rRNA processing and modification.

33© Oxford University Press 3136

A nucloolar protein with RNA binding motifs

A

B Strains

Redtwhits sectorlng at 23°C 32CC

NumbOr of mutants

++

/

Group I

-

2

Group 11

2+ 1

RW3 (starting strain)

+

RW 3

C s1136-w

sI136-w s1136-w p 36-6 (NOP77) ik.

s1136-w+ p136-6

iL

+

(NOP77)

230C

32CC

Fig. 1. Isolation of mutants exhibiting synthetic lethality with a mutation in the nucleolar protein NOPIp. (A) Schematic representation of the screening strain RW3. The nopl-5 allele was integrated in the genome of strain RW3 by homologous recombination with selection for the HIS3 marker gene. RW3 also carries plasmid pCH1122-URA3-ADE3-NOP1, which allows the formation of a red pigment in the ade2/ade3 genetic background. Growth of RW3 at 32°C without selection for the plasmid results in the formation of white sectors in the colonies, due to loss of the plasmid. After mutagenesis, synthetic lethal mutants were identified which are unable to lose the plasmid and therefore only form red colonies. (B) Growth characteristics of synthetic lethal mutants obtained from the genetic screen. SI mutants were grown on YPD plates for 5 days at 23°C or 32°C before red/white sectoring was scored. Of the sl mutants, 12 (Group II) exhibited red/white sectoring at 230C (+), but not at 320C (-). Group I sl mutants did not show red/white sectoring at any temperature. (C) Growth comparison of yeast strains RW3-w, s1136-w and s1136-w transformed with the complementing plasmid p136-6 (NOP77) at 230C and 320C. The construction of the strains is described in Materials and methods. Plasmid p136-6 which carries the wild-type NOP77 gene restores growth of s1136-w at 320C.

Results NOP77 is a novel protein which functionally interacts with the snoRNP protein NOP1 A mutant form of a NOPip-interacting component may exhibit an sl phenotype in combination with a ts allele of NOPip but allow viability if NOPip is intact. To screen for sl mutants, we used the nopl-5 allele, which is impaired in the production of both 18S and 25S rRNA at the restrictive temperature (Tollervey et al., 1993), in the ade2/ade3 based red/white colony sectoring assay (Koshland et al., 1985; Kranz and Holm, 1990; Wimmer et al., 1992). An ade2/ade3 screening strain was constructed which harbours an intact NOPI gene inserted into a URA3/ADE3-containing plasmid (pCHl 122-ADE3-URA3-NOPI) plus a mutated nopl-S allele which was integrated at the NOP1 genomic locus by homologous recombination (strain RW3). Strain RW3 carrying pCHl 122-ADE3-URA3-NOP1 is red; loss of the plasmid results in the formation of white sectors. Growth of RW3 on YPD plates at 32°C without selection for the plasmid resulted in red/white sectoring, showing that the nopl-5 product is still functional at this temperature, permitting the loss of the NOPJ-containing plasmid. Strain

RW3 was then mutagenized by UV irradiation. Among the -50 000 surviving colonies screened, 39 failed to show sectoring at 32°C. Of these, 14 regained a sectoring phenotype at 32°C when transformed with a plasmid carrying NOPI, but not nopl-5, demonstrating that these strains contain mutations which are synthetically lethal in combination with nopl-5. When these 14 mutants were grown at 230C, 12 of them exhibited weak red/white sectoring (Figure 1B, Group II). These sl mutants are therefore viable in the presence of nopl-5 at 230C but not at 320C. Complementation at 320C was used to clone the gene(s) responsible for synthetic lethality. Cells derived from a white sector (i.e. lacking the NOP] plasmid) of one of the 12 conditional sl mutants (s1136) grown at 230C, were isolated. The growth of these cells (s1136-w; w stands for white) was slow at 230C as compared with white cells derived from strain RW3 (RW3-w) and stopped at 320C (Figure IC). A yeast genomic library derived from a wild-type yeast strain JR26B x JU4-2 inserted into a low copy ARS/CEN plasmid (pUN100) was transformed into sl136-w and LEU+ transformants were selected for growth at 320C. Seven complementing strains were isolated; in 3137

T.Bergbs et al. A Table I. Yeast strains P65 I

Hind III

SphI

I

I

1*..-VPS16-._

TVA

ATO

1

615

901

Bgl11 :EcoR

g9i 11 Hind iN Ci I

Hind i l

I N*P77

-

Strain 2A1

*TA

2956

306

CH1462 RW3 B RNPL2 1 MEETIENVEV PSSNVSKQND DGLDMK¶LFV RS PQDVTDE QLADFFSNFA RNP-1

51 PIKHAVVVKD TNtKRRGFGF VS AVEDDTK EALAKARKTK FNGHILRVDI

RW3-w

s1136

101 AKRRDRSKKT SEVVEKSTPE SSEKITGQNN EDEDDADGED SMLKGKPII RNP-2 R--NPu 151 RNPWSCRD PVKLKKIFGR YGTVVEATIP RKRDGKIICGF AFVTKKISN 201 CRIALENTKD LKIDGRKVAV DFAVQKNRWE DYKKAQPEMN

tfW RNP-2

251 DAEENDDEE DENEEEDRQV DQASKNKESK RKAQNKREDF ' VPYD 301 ATEESLAAHF SKFGSVKYAL PVIDKSTGLA

RNP-1 KVAKD QYTYNECIKN

sl1 36-w

A136-3

351 APAAGSTSLL IGDDVMPEYV YEGRVLSITP TLVREDAGRM AEKNAAKRKE 401 ALGKAPGEKD RRNLYLLNEG RVVEGSKMAD LLTNTDMEIR EKSYKLRVEQ

ProtA-NSP1

451 LKKNPSLHLS MTRLAIRNLP RAMNDKALKA LARKAVVEFA TEVKNKERHP 501 LSKEEIIRST KEKYKFMGPD EIEAQKKKDK KSGVVKQAKV IMEVKGSTAG RNP 1 551 R RGYGFVED RHKNALMGL RWLNCHAVTS DEILEGLNDD EKKQVDNDLG 601 KGRRLCVEFA IENSNVVKRR REQLKQARTK RTRPDNEDTG DVGESENKKP

651

KKEEATrPTN PDDKKKGDDT KRTIGFKRKR KHAKK*

Fig. 2. Amino acid sequence of NOP77p predicted from its DNA sequence. (A) Restriction map of a 3.1 kb PstI-HindIll DNA restriction fragment which complements s1136-w. Adjacent to the NOP77 gene, whose ORF starts at position 901 and stops at position 2956, is the truncated VPS16 gene. (B) Amino acid sequence of NOP77p. The RNP-2 and RNP-1 sequences within the RRMs of NOP77p are boxed. Acidic amino acid stretches are underlined. A potential nuclear localization signal (NLS) at the C-terminal end of NOP77p is also underlined.

each case, growth at 32°C was shown to be dependent on the presence of the plasmid. Characterization of the complementing plasmids from these transformants showed that six carried the NOPJ gene. The plasmid not carrying the NOPI gene (p 136-6) was reintroduced into si 136-w and shown to restore growth at 32°C (Figure IC). When p136-6 was transformed into the remaining sl strains, it restored red/white sectoring to the other 11 which showed conditional synthetic lethality. Therefore, 12 of 14 sl mutants isolated can be complemented by a single gene. Plasmid p136-6 contains a genomic insert at 12 kb. The complementing activity was subsequently localized to a 3.1 kb PstI-Hindml fragment (Figure 2). On sequencing, two open reading frames were found; one was incomplete and corresponds to the C-terminal end of the VPS16 gene (Horazdovsky and Emr, 1993). The second open reading frame was complete, with an ATG start codon at position 901 and a stop codon at position 2956. The gene with the complete open reading frame was subsequently shown to contain the complementing activity for the s1136 strain (see also Figure 4). The open reading frame of the complementing gene encodes a novel protein of 686 amino acids with a predicted molecular weight of 77 kDa. Accordingly, we called the protein NOP77p. Sequence comparison of NOP77p with the EMBL data library showed a significant homology (35% identity within a 283 amino acid long overlap) to an incompletely sequenced open reading frame adjacent to the SW710 gene of Schizosaccharomycespombe (Rodel et al., 1992). Significant homology of NOP77p to several RNA binding proteins was

3138

ProtA-NOP77

Genotype MA Ta/a, ade2/ade2, his3/his3, trpl/trpl, leu2/leu2, ura3/ura3 MA4Ta, ade2, ade3, leu2, ura3, his3, can] MAMTa, ade2, ade3, leu2, ura3, HIS3::nopl-JS, can], pCH1 122-URA3-ADE3-NOP1 MAMTa, ade3, ade3, leu2,ura3, HIS3::nopl-J's, can] MATai, ade2, ade3, leu2, ura3, HIS3::nopl-Is, can], nop77-1Js, pCH1 122-URA3-ADE3-NOP1 MATat, ade2, ade3, leu2, ura3, HIS3::nop-5's, can], nop77-1Js MATa/a, ade2/ade2, leu2/leu2, trpl/trpl, ura3/ura3, his3/HIS3::nop77/NOP77 (derived from RS453) MATa, ade2, ade8, leu2, lysi, canl-100, URA3::nspl, his-, pSB32-LEU2-ProtANSPI (see Grandi et al., 1993) MATai, ade2, leu2, trpl, ura2, HIS3::nop77,

pUN100-LEU2-ProtA-NOP77 (derived from A136-3) GAL1O::ProtA-NOP77 MATa, ade2, leu2, trpl, ura3,HIS3::nop77, pUNIOO-LEU2-URA3::GAL1O: ProtANOP77 (derived from A136-3)

also found. In particular, four motifs matching the RNP-1 consensus sequence of RNA binding proteins (Bandziulis et al., 1989) are present in NOP77p (Figures 2 and 3). A closer inspection revealed that the entire consensus RNA recognition motif (RRM) which is -80 amino acids in length, including the highly conserved octapeptide RNP-1 and a more degenerate RNP-2 sequence, is found three times in the N-terminal half of NOP77p (Figure 3). A fourth imperfect RRM, lacking a clear RNP-2, is located in the C-terminal half of the protein (Figure 3). The amino acid composition of NOP77 reveals an organization into two domains of roughly equal size. An acidic N-terminal region (pl = 5.2) including the three consensus RRMs (residues 1-370) and a basic C-terminal region (pl = 10.6) including the less conserved RRM (Figure 3). The overall pI of NOP77p is 9.8. Identification of a critical amino acid residue within RRM3 of NOP77p involved in synthetic lethality with nop 1-5 The NOP77 allele present in strain s1136 is designated nop77-1. To clone and sequence the nop77-1 allele, a genomic sub-library was prepared from strain s1136 and the nop77-1 gene was isolated by colony hybridization (see Materials and methods). When introduced into strain s1136, nop77-1 failed to complement the sl phenotype (Figure 3C, upper panel). The entire nop77-1 gene was sequenced and found to contain a single amino acid substitution changing alanine-308 to proline as compared with the NOP77 allele cloned by complementation (Figure 3B and C, middle and lower panels). However, cloning and sequencing of the NOP77 allele in the parental strain RW3 revealed that this also contains a proline at position 308 (data not shown). The genomic bank used to clone NOP77by complementation was

A

-1t.rA

A nucleolar protein with RNA binding motifs 686

1

UIRRM 1 SSSS RRM 3 RRM 4 RRM 2

B

RNP-1

RNP-2

RRM I

P-QDTDEQLADPFSZ:F.?IKHN.-WKDT-NKRS RGFGFVSF

... LFVRS I

RRM 2

V

rbA-

r THrDT

L IIRNM PWSCRDP-V-KLKKIFGRYG-VVJEAT-IPRKR-ESKL CGFAFVTM KCKL

RRM 3

KGTAFVAF KDQYTYNECIKNAPAAGSTSLLIGDDVM. -J.VFVRNV P-YDY 3TEESLAAHFSKFGSV.YAt'DFVITDKSTGLA AT

RRM 4

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CONSENSUS C

LFVGNL IYIKG

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p

KE-KYKFMG PD EI EAQKKKDKKSG'vJvKQtAKVTsMEViKGSTzAGRS RGYGFVE F REHKNJ.LMG T R ;"Ti NCH.- T S E I LEG-LNJ.

A

({; X

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A1A

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PEb _iF Pi!i .RI- T-DA7E ESLAEF-r Cy; .-X k:.9WSIT

p Dpe rl'V RlN-0.A. E

K R

FG V Y I

F

NO.)V77 (pi 36-6)

N0P77

RHP-2

L

AFi AF7ix_; T-eluE: xNiAi A4:s' :_:s

;m

Fig. 3. NOP77 contains four RRMs. (A) Location of the four RRMs within the NOP77 amino acid sequence. (B) The amino acid sequences of the four RRMs in NOP77p were aligned according to the RRM consensus sequence, and RNP-1 and RNP-2 boxes are underlined (Bandziulis et al., 1989). Conserved residues are marked in bold. RRM4 lacks a typical RNP-2 box. The amino acid change Ala3O8 Pro nop77-1 maps within RRM3 and is shown with an asterisk. (C) Growth comparison of yeast strain s1136 transformed with the complementing plasmid (NOP77) and non-complementing plasmid (nop77-1) at 32°C on 5-FOA-containing plates. In each case, two individual transformants are shown (upper panel). DNA sequence and deduced amino acid sequence of the NOP77 and nop77-1 alleles of the region surrounding Ala/Pro3O8 (middle panel). Predicted secondary structures RRM3 of NOP77p and nop77-lp according to the algorithm of Chou and Fasman (1978). The dotted line shows the probability for a-helical folding; the solid shows the probability of folding in a-sheets. The proline in nop77-lp is predicted to truncate a-helix al -

RRM3/nop77-1

RNP-1

I AL.:;S IF 5V X VtL P V: 7STQ' IL 3TA FV9 PXCItYG 12 XICA P). A'

"

-IF %;-,.e ".Nr

s5L'.

si .;>

-.r

derived from another wild-type yeast strain (JR26B x JU4-2) and the Ala308 to Pro3O8 substitution is therefore the result of a natural polymorphism. In another laboratory strain of Saccharomyces cerevisiae, the NOP77 gene also contains proline at position 308 (J.Woolford, personal communication). It appears that a mutation in the s1136 strain alters the genetic background such that the Pro3O8 allele (nop77-1) becomes conditionally sl in combination with nopl-5, while the Ala308 allele (NOP77) is not. To confirm that the Pro3O8 allele is silent in a NOPI + genetic background, the nop77-1 gene was expressed in a

(lower

panel).

nop77::HIS3 null strain. No growth difference relative to a NOP77 strain was observed at any temperature tested (23°C, 30°C or 37°C) (data not shown). Interestingly, this amino acid polymorphism maps in the third RRM of NOP77p, affecting the predicted secondary structure of ahelix ao1, which is located between RNP-2 and RNP-1 (Figure 3C, lower panel). The synthetic lethality between nop77-1 and nopl mutations is not allele specific for nopl-5; synthetic lethality was also found with other ts nopl alleles as shown by transforming s1136 with plasmids containing nopl-3 and nopl-7alleles (see also Materials and methods). 3139

T.Bergbs et a!. A

ProtA-NOP77

z

T

I. 130

B

NOP 77 -

-___

686

c

anti-ProtA r 106 80

496S

Fig. 4. Tagging of NOP77 with IgG binding sequencess derived from S.aureus protein A. (A) Schematic drawing of the ProttA-NOP77 fusion protein. Two IgG binding domains (130 residue S.aureus protein A were fused in-frame to the N-termi inal end of NOP77p (686 residues). Transcription of the ProtA -A {OP77 gene is under the control of the NOPI promoter. (B) The Prot LA-NOP77 fusion protein is functional in yeast. When the diploid strain A 13623 (NOP77/nop77::HIS3) was sporulated and tetrads were dissected, a 2:2 segregation for viability was observed (upper panel). VVhen A 136-3 was transformed with plasmid pUN100-Leu2-ProtA-N( )p77, HIS+/LEU+ progeny could be recovered after tetrad d lissection indicating that expression of ProtA-NOP77 fully comj plements the otherwise lethal nop77::HIS3 mutation (lower panel). Qc) Expression of the ProtA-NOP77 fusion protein in yeast. A whole cell extract was prepared from a haploid strain expressing the ProttA-NOP77 fusion protein (nop77::HIS3 plus pUNIOO-LEU2-ProtA L-NOP77) and analysed by Western blotting. The blot was decorated with IgG coupled to horseradish peroxidase allowing detection or IfProtA-NOP77 (indicated by an arrow).

NOP77 is a nucleolar protein essential for cell growth To analyse the in vivo function of NOP77, wie disrupted its gene by replacing a 1 kb BglII restriction fhagment within the open reading frame of NOP77 (residiue 166-508) with the HIS3 gene. The nop77::HIS3 c-onstruct was integrated at the NOP77 genomic locus in a 4diploid strain. Correct integration and gene replacement we re verified by Southern analysis (data not shown). Tetrad anialysis showed a 2:2 segregation of cell viability (Figure 41B), the viable progeny being always NOP77+. When the diploid strain heterozygous for NOP77 was transformed M vith a plasmid containing the cloned NOP77 gene and sporulated, a 4:0 segregation for viability was observed I and haploid nop77::HIS3 progeny always contained the N'OP77plasmid (data not shown). This demonstrates that NOP '77 is essential for cell viability. To determine the subcellular localization c)f NOP77p, a tagged fusion protein consisting of two IgG bin(ding sequences from Staphylococcus aureus protein A fused to the N-terminal end of NOP77p was expressed in yeast (Figure 4A). Tagging of proteins with protein A seqi uences, which bind with high efficiency to IgG molecules, iE a convenient method to immunolocalize fusion proteins wit thin yeast cells (Wimmer et al., 1992; Grandi et al., 1993; Jansen et al., 1993). Haploid nop77::HIS3 cells grow norrmally if they express the ProtA -NOP77 fusion protein, slhowing that it is fully functional (Figure 4B). Whole cell extracts were

3140

prepared from such a complemented strain and analysed by Western blotting using IgG-horseradish peroxidase to detect the protein A moiety. A band of 110 kDa, slightly larger than the calculated molecular weight of 95 kDa, was detected by immunoblotting (Figure 4C). Indirect immunofluorescence was performed with cells expressing the ProtA-NOP77 protein. A cap-like nuclear staining typical of nucleolar localization was observed in most cells (Figure 5, upper left panel; see also Schimmang et al., 1989; Aris and Blobel, 1991; Jansen et al., 1993). When double immunofluorescence was performed to detect ProtA NOP77 (red) and NOPip (green), the fluorescence signals fully overlapped, resulting in yellow fluorescence emission (Figure 5, upper right panel). Staining of ProtA-NOP77 (red) and DNA (blue) revealed that the bulk of the chromatin was generally opposite ProtA-NOP77, again indicating a nucleolar localization (Figure 5, middle right panel). Immunoprecipitation from a strain expressing the ProtANOP77 fusion was used to test for a physical association between NOP77p and NOPlp or the essential snoRNPs. About 20% of the ProtA-NOP77 could be immunoprecipitated from a cell lysate prepared under non-denaturing conditions, using IgG coupled to ProtA-Sepharose (data not shown). Proteins co-precipitated with ProtA -NOP77 were analysed by Western blotting. NOPip was detectably but inefficiently precipitated (