component ofa larger protein; other modules in such proteins include an ATP-binding motif, a zinc-binding finger, and

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Proc.Nati.Acad. Sci. USA Vol. 88, pp. 2495-2499, March 1991 Biochemistry

RNA-binding domain of the A protein component of the U1 small nuclear ribonucleoprotein analyzed by NMR spectroscopy is

structurally similar to ribosomal proteins

(NMRstructure/ribonucleoprotein consensus sequence/RNArecognitionmotif)

DAVID W. HOFFMAN, CHARLES C. QUERY, BARBARA L. GOLDEN, STEPHEN W. WHITE, ANDJACK D. KEENE*

Departmentof Microbiology and Immunology, Duke University Medical Center, Durham, NC 27710 Communicatedby W. K. Joklik, December 21, 1990 (received for review November 15, 1990)

ABSTRACT An RNA recognition motif (RRM) of '480 amino acids constitutes the core of RNA-binding domains found in a largefamily of proteins involved in RNA processing.

TheUlRNA-binding domain of the A protein component of the human Ul small nuclear ribonucleoprotein (RNP), which encompasses the RRM sequence, wasanalyzed by usingNMR spectroscopy. The domain oftheAprotein isahighly stable monomer insolution consisting of fourantiparallel f-strands and two a-helices. The highly conserved RNP1 and RNP2 consensussequences,containing residues previouslysuggested to be involved in nucleic acid binding, are juxtaposed in adjacentf-strands. Conservedaromatic side chains that are critical for RNAbinding are clustered on the surface of the moleculeadjacent to a variable loop that influences recognition ofspecific RNAsequences. The secondary structure and to- pology ofthe RRM are similar to thoseofribosomalproteins L12 and L30, suggesting a distant evolutionary relationship between these twotypes ofRNA-associatedproteins.

Over the past decade, the ways in which proteins interact specifically and nonspecifically with DNA have been revealed bybiochemical and biophysical studies (reviewed in refs. 1and2). Muchof this information has been the result of crystallographic analysis, but more recently, NMR tech- niques have been used (3). As regardsRNA-binding proteins, however, there has been littlestructural information with the notableexceptionof the work by Steitz and coworkers (4)on theglutaminyl-tRNA synthetase/tRNAGIn complex.

Sequence elements characteristic of a group of RNA- associatedproteins begantoemergewith theobservationof fourcopies of an 80-amino acid repeat in poly(A)-binding protein(5). Themost conserved region of8 amino acids in theserepeats wastermed the ribonucleoprotein(RNP)consensus sequence, or octamer (RNP1) (6). Another 6-amino acidregion waslater termedRNP2(7).It wassubsequently shownbyseveralgroupsthatthesequencesimilarityappears in many other RNA-associated proteins (7-11) (recently reviewed in ref. 12), containsmanyconserved positions, and isnotably rich in aromatic residues (8). Direct evidence ofan interaction between this motif and RNA has led to its designationasanRNArecognition motif, or RRM(8). This motif has also been referred to as an RNP consensus sequence-type RNA binding domain (11) and as an RNP-80 motif(13). Members ofthe RRM-containing family ofproteins include heterogeneous nuclear RNP proteins and U-class small nuclear RNP (snRNP) proteins, as well as poly(A)-binding protein,nucleolin,transcription termination factorsLa and rho, eukaryotic initiation factor 4B, and sex- determination proteins in Drosophila. The sources of these

proteins range from Escherichia coli to man; thus, the RRM isclearlyanancient proteinstructure.

The RRM can be viewed as analogous tothe homeobox domain (5)or tothe zinc-binding finger motif (14) of DNA- binding proteins. In many cases, the RRM is a modular componentofalargerprotein; other modules in suchproteins include an ATP-binding motif, a zinc-binding finger, and oftenone or moreadditional RRMs (12). It has beenpostu- lated that, insome cases, thepresenceofmultiple RRMs in aproteinmayallowforbridging betweentwodifferentRNA molecules (15).

We have shownpreviously that the RRM constitutes the core ofa U1 RNA-binding domain for the 70K (52-kDa) protein of U1 snRNP (8). In addition, the A proteinof U1 snRNP(UlsnRNP-A)containstwo RRMs,and subsequent studies demonstrated that the N-terminal RRM alone constitutes theRNA-binding domain for stem-loop II (nucleotides 49-85) of U1 RNA (13, 15). In ordertounderstand the structure and function of these RNA-binding domains in greater detail and to enable better designed mutagenesis studies,wehaveused NMRspectroscopy tocharacterizethe U1RNA-bindingdomainofthe UlsnRNP-Aprotein. From ourstructuralcharacterization,wehavededucedamodel of the RRM thatexplains manyof itsexperimentally observed features and identifies its probable site ofinteraction with RNA.

MATERIALS AND METHODS

Purification oftheUl RNA-BindingDomain of the A Pro- tein.The U1RNA-bindingdomain from the N terminus of the UlsnRNP-A protein was expressed in E. coli BL21 as a fusionproteinwithglutathione S-transferase (16).After cell lysisandammonium sulfateprecipitation, thefusionprotein was purified on aglutathione-agarose affinity column and cleaved with thrombin(Sigma) overnightat370C.Finally,the releasedglutathioneS-transferase and residual fusionprotein were removed by using S-Sepharose column chromatogra- phy. The purified RNA-binding domain contained residues 11-96ofU1snRNP-A plus four additional amino acids(Gly- Ser-Met-Gly)attheNterminus. These residues didnotaffect the binding activity of the domain. Residues 11-96 were demonstratedpreviouslytoconstitutetheminimalU1 RNA- bindingdomainhavingthe sameaffinityasthefull-lengthA protein (15).

Proteinconcentrationwasdeterminedbyabsorptionmea- surements at280nmbyusinganextinctioncoefficient of5520 M-1-cm-lasdeterminedbyquantitativeamino acidanalysis.

The 15N-labeled U1 RNA-binding domain was prepared by

Abbreviations:RRM, RNArecognition motif;RNP,ribonucleoprotein; snRNP, small nuclear RNP; VR1, variable region 1; NOE, nuclearOverhausereffect;UIsnRNP-A, the Aproteinof U1 snRNP.

*To whomreprintrequests should be addressed.

2495 Thepublicationcostsof this article weredefrayedinpart by page charge payment.This article must therefore beherebymarked "advertisement"

inaccordance with 18 U.S.C. §1734 solelytoindicate this fact.

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2496 Biochemistry: Hoffman et al.

growing the overproducing strain of E. coli on minimal medium plus nucleotides at 40 mg/liter supplemented as follows. For

'5N-labeled

glycine and serine, [15N]glycine (Cambridge Isotope Laboratories, Cambridge, MA) at 100 mg/liter,noserine, and the other amino acids at 200mg/liter were added.For15N-labeled leucine,isoleucine, and valine, [15N]leucine(MSD Isotopes) at 100 mg/liter, no isoleucine or valine, and the other aminoacids at 200mg/literwere added.

For 15N-labeled lysine, [15N]lysine (MSD Isotopes) at 30 mg/liter and the other amino acids at 200 mg/liter were added.

NMR Spectroscopy. NMR samples typically contained 3 mMprotein equilibrated in4 mMacetic acid at pH 3.1 and 34°C. Spectrum assignments were made by using two- dimensional nuclear Overhauser effect (NOE), two- quantum-filtered correlated spectroscopy (17), relayed co- herence (18), and Hartmann-Hahn correlated (19) spectra.

For samples in 2H20, the signal from residual, H20 was suppressed by low-powerpresaturation. Forsamples in95%

H20/5%2H20,theH20suppression wasperformed using the SCUBA(20)pulsesequence. Proteins containingamino acids specificallylabeled with 15N were used in

1H-15N

heteronu- clearmultiple-quantumcoherenceexperiments (21) to iden- tify the amide protonsofthelabeled amino acids (22). Spectra wereobtained using aGeneral Electric GN-500 spectrometer at 500 MHz, except for the Hartmann-Hahn correlated spectra, which wereobtained on aBruker AMX spectrometer at600 MHz. NMRdatawas processed by usingFELIX software byHare Research, Woodinville, WA.

RESULTS

StructureDetermination and Modeling. The NMR spectra of the RNA-binding domain of the UlsnRNP-A protein provided evidence of a stable, well-defined structure. For example, thetwo-quantum-filtered correlated spectroscopy spectrum (Fig. LA) shows many resonances that were sig-

A

LF)rr)

0

IT}

E

0

Ln)

LU)

9.5 9.0 8.5 8.0 7.5 7.0

(ppm)

nificantly shifted away from the random-coil spectral positions. Also, 18 backbone amide protons remained unex- changed when theprotein was dissolved in2H20.Asshown inFigs. 2 and 3, these are either involved in stablehydrogen bonds or are buried in the hydrophobic core. The NMR linewidths are typically 6-9 Hz, as expected of a 1O-kDa protein, indicating that the N-terminal RRM of the UlsnRNP-A protein is a monomer in solution. Spectra at variouspH values and temperaturesshowedthatthe structural integrity ismaintained at conditions that are ideal for NMRexperiments (pH range of 3-7 and temperatures upto 500C).

Two-dimensional NMR methods were used toassignres- onances tospecificprotons in the U1RNA-bindingdomain.

A total of 364 distinct proton resonances were assigned, includingall except 2oftheresidues betweenresidues11and 93 in thesequence. The assignment problem was simplified by heteronuclear NMR experiments on the three protein samples containing selected

'IN-labeled

amino acid types.

Thesequential NOE data show patternsconsistentwithtwo segments of a-helix and four segments of p-strand, in a

3-a-p-P-a-P

pattern(Fig. 2). The a-helices weredefinedby stretches of NOEsbetween the amide protonsofsequential residues(Fig. 1B) and residues i to i+3. The B-strandswere identified by strong sequential amide-Caproton NOEs and weak intraresidue amide-Ca NOEs. The pattern of inter- strandNOEs andhydrogen bonds(Fig.3) shows thatthe U1 RNA-bindingdomain contains a well-defined four-stranded antiparallel 8-pleated sheet. Strand /32 contains a bulge between residues 41 and 43. NOE patterns consistentwith tightturnsoccuratthebeginningof each of the twoa-helices.

Residues 46-49, 73-81, and 88-93 are involved in secondary structures that we are, as yet,unable to characterize from the NOE patterns. The three amino acids at each terminus were apparently disordered based upon the lack of associated NOEpeaks.

B

E a.

9.0 e ⁵ ^8.0

(pPMr

FIG. 1. Regionsoftwotwo-dimensional NMRspectraof the UlRNA-bindingdomain of the U1snRNP-Aprotein demonstrating amino acid assignmentsandaregionof a-helix.(A) Fingerprint regionofthetwo-quantum-filtered correlatedspectruminH20 solvent showing cross-peaks betweenamide and C'protonsbelongingtothe^sameaminoacidresidue. Allamideprotonswereassignedtocross-peaks with the exception of thethree aminoacidsateach terminus. Selectedcross-peaks^arelabeled accordingtothe one-letter code for amino acids, and their numbering correspondstothatof thefull-length protein(23).Unlabeledcross-peakshave beenassignedorareaccountedforbytheterminalamino acids.

(B)Asection ofthe 200-msec NOEspectrumofthe UlRNA-binding domain. NOEs between thepairs of sequential amideprotonsthat form helixalarelabeled.

V62 L69 A65

Y78 K y3 K27

oc T66 I93 R70 I212

I40 K22 L2 KOg K60 036

L29 8PM82 U-

E41 L4 vD24@ N16 F7 F37

1141 1 .4'92N6. F

L443 Y86 I1433 M72 4 4

L444

³ ^R83

3s viyI43E6 F75

F593 112w EI.F5

N15 A55

II 054

J~~~~~~~E FF56

Proc. Natl. Acad Sci. USA 88

(1991)

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Proc. Natl.Acad. Sci. USA 88 (1991) 2497

I I 15 2do 25 30 35

T IY IN N

E]N

^{E K} ^I K K D E L ^{K K} S L A I FQ

E

^G

.4- 03! s-o

-C

I -B

000 0@* *0 6

__________- _

0000 00 00

40 45 50 55 60 65

QWE]L

^D ^I ^L ^V ^S ^R ^S ^[L ^K ^R G. Q .A F V I F K E V S

SW]T

.4- 52 - .4 3 - _ - a2-

6 0

- -

0 6 .0 .0

_ - _~~-* *-

= . I*

70 75 80 85 90

N AL R SEmQ[E]F

P[E]Y[E]K P[H]RWm

^Q ^Y f K T[]S D I I .4---- 4 - 0

U_

0

U-

FIG. 2. Summary ofsequencehomology and NMR data for theUlRNA-binding domain of the UlsnRNP-Aprotein.Residues^arenumbered

tobeconsistent with the full-length U1snRNP-A protein (23) and labeled accordingtothe one-letter codeforamino acids.Boxedresiduesare

conservedamongtheRRM-containing proteins; stippled residuesareconservedfor structuralratherthan functionalpurposesandaremainly in thehydrophobiccore. Filledcircles indicate backbone amideprotonswithexchange timesgreaterthan 24 hr indeuterated solvent.Bars indicateobserved sequential NOEs.Inthecaseof amide-amide(NN)andamidedCa(aN) sequentialNOEs, the bar thicknessis proportional totheapproximateNOEintensity.Open diamonds indicateaNOE from residueitoi+3.NOEs thatarenotobservable duetoamideproton chemical shiftdegeneracyaremarked with^anasterisk. Residues 52-59 constitute RNP1 and 12-17 constituteRNP2.

The NMR results provide detailed information on the complete secondary structure of the molecule inwhich the approximate positions of the side chains could be determined. Byusingcomputergraphics, itwaspossibletodock these secondary structural elements ina waythat is consistent with the positions of hydrophobic core residues, the location of the tight turns, and the lengths of the intercon- necting loops. The probablehydrophobiccoreresidues (see Figs. 2 and4)werededuced from the alignment ofthemany

RRM sequences that are now known (ref. 8; D. Kenan, C.C.Q., andJ.D.K., unpublished results). This initial model

wasfurtheradjustedso astobe consistent with theobserved NOEs between nonsequential amino acids. These NOEs included, among others, signals between the side chains of Val-57 and Tyr-31, Leu-69 and Tyr-86, and Ala-68 and Tyr-86. This generalprocess can be considered amodified version of the "combinatorial approach" first described by Cohen and Sternberg (26).

Description of the Molecule. A schematicrepresentation of themolecule is shown inFig. 4. It belongsto thea/(3 class ofproteins (27) and consists oftwo helices packed on the

same side ofa four-strand antiparallel (8-pleated sheet to produce a helix-sheet, two-layered molecule. Several features of the model support its general validity. First, the

(3-a-(3 connections are both right-handed, and this has

proven to be one of the least violated rules of protein structure (28, 29). Second, the general pattern ofproton- deuterium exchange shown in Fig. 2 agrees well with the exposed and buried regions of the molecule. Finally, oneof themosthighly conserved residues in the RRM is Ala-68 (8), and theCaprotonof this residue exhibits^anunusualchemical shift of 2.18 ppm. Inthe model, this residue is completely buried andcloseto twoaromaticrings.Thehydrophobiccore

of the domain consists of residues Ile-12, Ile-14, Ile-40, Ala-55, Val-57, Phe-59, Met-82, and Ile-84 ^on the inner

surface of thefour-strand sheet (a3, /32, (33, and,(4);residues Leu-26, Leu-30, and Phe-34onhelixal; and residues Ala-65, Ala-68, Leu-69, and Met-72on,(2.The exposed surface ofthe sheet contains residues that are eitherconsistently hydro- philic orthathave been shown in another RRM-containing protein tocross-link to bound nucleic acid (10). The latter (residues Tyr-13 andPhe-56)are indicated inFig. 4.

Extensiveamino acidanalyses of all RRM sequences (D.

Kenan, C.C.Q., and J.D.K., unpublished results)using the multiple alignmentprogramTULLA(30) and secondarystruc- turepredictive algorithms indicate that the three-dimensional structuredescribed above ishighly conserved in this family.

The most variable region is the loop between (2 and (83 [variable region 1 (VR1)],which is knowntocontain elements that determine RNA binding specificity (see below). This region,however,appearsnottoaffect the overallstructureof themolecule.Forexample,amutantprotein with five amino acidchangesin thisregion hasanaltered RNAspecificity (24) butdisplaysanNMRspectrumsimilartothat of the wild-type domain (data not shown). The RNP1 octamer is highly conservedbecauseitcontainsbothstructurallyand function- ally important residues. The structural residuesinclude the hydrophobic core amino acids 55, 57, and 59, which are

distinct from the conserved aromatic residues 54 and 56on

the surface, which are presumed to be involved in RNA recognition.

DISCUSSION

Implications for RNA Binding. The NMR analysis has revealed thatmanyof the conserved residues areimportant for structuralintegrity, whichisconsistentwith theirhydro- phobic character. On the other hand, other conserved resi- duesareclusteredonthe surface of the (3-pleated sheet and, therefore, areavailableforbindingtoRNA.Theseresidues

N %0,itIi ) tN(i i+l-) N(i.i+1) caNti. -3}

-I1:0 NNlti+l-) aN0i,i+l) SN{I,i+l) aN Oi. i+31

-1 -(.1 NN(i,i+1) aN (i,i-4CI) DN{i,i-I) aN (i.i+3)

Biochemistry:

Hoffman etal.

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2498 Biochemistry: Hoffman et al.

0 H H O H H 0 H

CCNCCNCC

C C N ^HO ^H N C C

H 8 H O

FL

⁴

H 0Ro H ®L110 H 0 H

C N C C N C C N

-%-_ C_ _C_ _g-_^_C_ ^_C_ ^_ ^C_

C N C C N C C N C

H (i) SO ) H

H 0 H 0 H 0

C N CC N, C.00_N CC_ ^C

0 H H 0 H 0 H H

H H H 0

C N C C N C

N C C N C C N

H 0 H H 0 H H

FIG.3. 3-Sheetstructureof the U1RNA-binding domain of the UlsnRNP-Aprotein. Cross-strand NOEsareindicatedbyarrows;

amideprotonsprotected from solvent exchangearecircled; hydro-

genbondsareindicatedby dotted lines. The amino acidsarelabeled accordingtothe one-lettercode, and their numbering correspondsto that of thefull-length protein (23). Residues Leu-41toIle-43repre- sentabulge in the 13-sheet. Note the juxtaposition of Phe-56 in the RNP1octamerand Tyr-13 inRNP2, both of which projectawayfrom thehydrophobiccoreof the domaintothesurface. Corresponding residues in the hnRNP-A1proteinwereUVcross-linkedtooligo(de- oxythymidine) (10).

areTyr-13, Gln-54, andPhe-56. Severalindependent results supportthis idea.

First,it has beendemonstratedthat theresiduesequivalent to Tyr-13 and Phe-56 in the Al protein of heterogeneous nuclear RNP can beUV cross-linked to oligo(deoxythymi- dine) (10) andmusttherefore be closetothe bound nucleic acid.Second, mutagenesis studiesonthe 70Kprotein of Ul

FIG. 4. A schematicdiagramof theU1RNA-bindingdomain of the UlsnRNP-A proteinas determinedbytwo-dimensional NMR and combinatorial model building. 13-Strands are represented as arrows,and the a-helices^arerepresented^ascylinders.Residuesare

numbered to be consistent with thefull-length UlsnRNP-A protein (23) andlabeledaccordingto theone-letter codefor amino acids.

Residuescorrespondingto those inheterogenousnuclear RNP-A1 proteinthat have been shown to cross-link to boundnucleic acid (Tyr-13andPhe-56) areindicated in thediagram. Thehighlycon- served RNP1 octamer sequence is hatched forclarityandconsists of strand 133 and residue R52. RNP2consists of strand 131 andresidue Leu-17. Loop3 contains amino acidsthathelptodetermine RNA- binding specificity (24, 25).

snRNP and 60-kDa Ro protein have shown that the phenylalanine or tyrosine residue, respectively, at the corresponding position 56 is absolutely required and that the corresponding positions 13 and 54 require aromatic residues (C.C.Q., S. L. Deutscher, and J.D.K., unpublished data).

Theglutamine at position 54 in UlsnRNP-A protein is an unexplained anomaly in this respect. It should also be noted thattheconserved Arg-52 in theRNP1octamerisadjacentto this group of aromatic residues and available forbindingthe nucleic acid (Fig. 4). In addition, other basic residues at positions 20, 47, 50,80, and 83 arein this vicinity and may alsoparticipate in interactions with RNA.

Sequenceelements withinthe RRM that determine binding specificityarenotunderstood. However, in at least one case, asmall regionin the center of themotifhasbeen shownto affectbinding specificity.UlsnRNP-Aand U2snRNP-B"are homologous proteins that bind to stem-loop regions of Ul (stem-loop II) and U2 (stem-loop IV) RNAs, respectively.

The loop regions of these RNAs appear to contain the specificity-determiningelements, despite having very similar sequences(25). Bentley and Keene (24) and Scherlyetal.(25) have independently shown that the specificity ofAand B"

proteinscanbe acquired by oneanotheruponexchangeofa polypeptide segment (residues 44-48 of A protein). This regionhasbeen calledVR1andoverlapsloop 3. Itsproximity to the conserved aromatic residues in RNP1 and RNP2 is evident inFig. 4. It isthereforelikelythat, in at least these two proteins, residues in this region recognize bases in a single-strandedRNAloop.

Atthe present stageof the structural analysis, it is prema- ture toproposedetailed models for the interaction with RNA.

However, some striking similaritiestoanother classofpro- teins that bindsingle-stranded nucleic acids, and for whicha model has been proposed, are evident. This class isexem- plified bygene 5 proteinfrom bacteriophage fd,and amino acid sequenceanalyseshave revealedseveral othermembers (31). The x-ray structureofgene 5 protein (32)shows that, like theRRM,conservedaromatic residues arepositionedon thesurface ofa

.-pleated

sheet. Resultsfrom NMR,chemical modifications, and cross-linking demonstrate the involve- mentofaromaticresidues in thebinding ofsingle-stranded DNA (reviewed inref. 1). Model building withthe gene 5 proteinand single-stranded DNAconfirmed that this could occur by aromatic ring-stacking interactions between the bases andtyrosineand phenylalanine sidechains (33).

RibosomalProteinSimilarity.Thehelix-sheettwo-layered structureofthe RRMis reminiscent ofthestructuresoftwo prokaryotic ribosomal proteins, the C-terminal halfofL7/

L12(L12CTF) (34)andL30(35).Thesimilarityisevenmore strikingin that the orderofthesecondarystructural elements within the sequences and theirtopological arrangements in thestructures arealmostidentical. As shown inFig. 5,taking the smaller L30asthebase,itis clear thataloop betweenal and82 isreplaced byahelix inL12CTF anda(-strand(132) in the RRM. It should be noted also that, although the ribosomalproteinshaveno RRM-like aromaticresidues on the sheet surface for potential RNA interaction, L30 does haveagroupof conserved polar and charged residues in the equivalentposition. These residues have beenpostulatedto constitute a-site of interaction with ribosomal RNA(35).

Adetailed structuralanalysis of the tworibosomal proteins concluded thattheirstructuralhomology isaresultof diver- gent evolution (36), and subsequent amino acid sequence comparisons of the ribosomal proteins indicated that their structural motif may be present in otherribosomalproteins (S.W.W., unpublished results). Is itpossible thattheRRM structuralsimilaritytothisribosomalproteinmotifreflectsan

evolutionary relationship? It is notunreasonable to expect that the RNA-protein complexes that orchestrate nuclear mRNAsplicing(thespliceosome)andproteinsynthesis (the

Proc. Natl. Acad. Sci. USA 88

(1991)

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Proc. Natl.Acad. Sci. USA 88 (1991) 2499

RRM

c

ribosomal

protein

L30

N

ribosomal protein L7/L12

FIG. 5. Secondarystructuralcomparisonof theU1 RNA-binding

domainof the U1snRNP-A proteindeterminedbyNMR spectros- copywiththat of ribosomal proteins L30andL7/L12 determined previously byx-raycrystallographic analysis (35, 36).Thediagram

shownasthe RRM representsthegeneral structuralfeaturespre- dicted for all members of the RRM family based upon primary sequencehomologies (8).Thecircles andtrianglesrepresenta-helices andP-strands, respectively.Ifthepointof thetriangleisupward,

thestrand isorientedsuch that the N terminusistoward thereader.

Helices and strands arenumbered to be consistent withthe RRM structure.

ribosome) may be evolutionarily related. Interestingly, ^a functional link between these processes is provided by eu-

karyoticinitiationfactor2,whichwasrecentlyshown to be requiredfor splicingin vitro (R. Padgett, personalcommu- nication). Furthermore, eukaryotic initiation factor4B was

recentlyidentifiedasamember of the RRMfamily(37),and poly(A)-binding protein is also required for initiation of translation (38). Itshouldbenoted thatacomparisonof the amino acidsequences ofRRMsand the ribosomalproteins L30andL12CTFshowednosignificanthomology.However, it is wellknownthatprimarystructureisconservedtoafar lesser extentthantertiary structure, andaputativecommon

ancestorwould have beenatrulyancientprotein.Finally,a

caveatto thishypothesisistheenzymeacylphosphatase (39), which has a very similar fold to the RRM, ^as suggested previously (40), but noapparent association with RNA.

This paper is dedicated to the memory of Professor Helen R.

Whiteleyfor herlove of science andhercouragein life.We thank DanKenanfor theTULLAanalyses,KimberlyWoodsforassistance with protein purification, and Stephen Brown for assistance in obtainingtheHartmann-Hahnspectra.Thisworkwassupportedby

researchgrantsfromthe NationalInstitutes of Health andthe Pew

Scholars Program to J.D.K. B.L.G. was supported by Cell and MolecularBiology TrainingGrantGM07184.

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Biochemistry:

Hoffman etaL