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nature structural biology • volume 7 number 4 • april 2000 263

RNA–protein interactions play centralroles in the regulation of gene expres-sion1. In eukaryotes, proteins bind tonascent mRNAs to control their process-ing and export to the cytoplasm, and theireventual translation into protein. Howprotein–RNA interactions exert theircontrol over these processes has remainedsomewhat a mystery. Nevertheless, onewell-studied example, the U1A protein,has given some answers. The U1A proteinregulates its own translation by bindingto an RNA element, called the poly-adenylation inhibition element, PIE, inthe 3' untranslated region of its mRNA2–4.A biomolecular complex of U1A proteinon the mRNA inhibits the activity ofpoly(A) polymerase (PAP), leading to adecrease in translation of the U1A protein(Fig. 1). On page 329 of this issue ofNature Structural Biology, the structure ofthe U1A dimer bound to its target RNAhas been solved by a tour de force NMRstudy5. The results of this work explainthe cooperative inhibition of poly(A)polymerase by the U1A protein.

The U1A protein contains an RNA-binding domain. U1A protein is a com-ponent of the U1 small nuclearribonucleoprotein complex (U1 snRNP),which is one of five snRNPs that make upthe splicing machinery (spliceosome) andis essential for splicing of mRNAs. Thecrystal structure of the RNA bindingdomain of the U1A protein6 revealed afour-stranded β-sheet that creates a bind-ing surface for RNA, with two α-heliceson the ‘back’ side of the protein structure,which stabilize the overall protein fold.Subsequently, the crystal structure of theRNA-binding domain of the U1A proteinbound to a 21-nucleotide RNA hairpinderived from U1 snRNA showed how theRNA binding domain of the U1A proteinrecognizes RNA7. Individual nucleotideswithin the RNA hairpin loop lie along thesurface of the β-sheet and are unstacked.Specific hydrogen bonds and electrostaticcontacts are made between amino acids

in the RNP motif and the bases and back-bone of the U1A hairpin loop. Thesplayed-out disposition of the bases isstabilized by extensive stacking andhydrophobic interactions with aromaticand aliphatic amino acid side chains.

Varani and coworkers previously solvedthe structure of a single U1A proteinRNA-binding domain bound to the 3'-untranslated region (UTR) of its pre-mRNA, which is an asymmetric internalloop with a sequence similar to the hair-pin loop in U1 RNA8. The nucleotides inthe internal loop of this regulatory regionare recognized by the U1A protein in amanner that is similar to that observed inthe hairpin–U1A complex7. In both cases,

the extensive network of RNA–proteincontacts leads to high-affinity (Kd ~nM)binding.

Binding of the U1A protein to the 3'UTR ultimately leads to inhibition ofpoly(A) polymerase. How is thisachieved? In this issue, Varani andcoworkers5 present the structure of twoU1A molecules bound to PIE RNA andprovide biochemical results to addressU1A protein function. PIE RNA containstwo asymmetric internal loops separatedby four Watson-Crick base pairs. Varaniand colleagues5 performed binding assaysto measure the interaction of U1A proteinwith PIE RNA, using gel mobility shifts,and examined the ability of different RNA

mRNA processing: The 3'-end justifies themeansJoseph D. Puglisi

The U1A protein regulates the activity of poly(A) polymerase by interacting with an RNA element in the 3' untranslated region of its mRNA. The structure of the trimolecular complex made up of two U1A moleculesbound to the target RNA explains the cooperative regulation in this system.

AAAAAAA5' 3'

AUUGC

AC

A

A U UG

CAC

A

PIE

AAA5' 3'

AUUGC

AC

A

A U UG

CAC

A

PIE

U1A

U1A

PAP

PAP

Fig. 1 Schematic of the 3' untranslated region (3' UTR) of the U1A pre-mRNA. The double internalloop of the PIE RNA element is shown explicitly (top). At low concentrations of U1A protein, thereis no binding of U1A to PIE RNA, and poly(A) polymerase (PAP) is not inhibited (top). At higherconcentrations of U1A protein, there is cooperative binding of two U1A proteins to PIE RNA; thedimer subsequently interacts with poly(A) polymerase, inhibiting its activity (bottom).

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constructs to stimulate polyadenylationin vitro. They find that two protein mole-cules bind cooperatively to PIE RNA;changing the spacing between the twoasymmetric loops by increasing ordecreasing the number of base pairs dis-rupts the cooperativity of binding. Theauthors also show that activation ofpoly(A) polymerase requires two U1Aproteins bound to PIE RNA to activatepoly(A) polymerase. These findings havea number of structural implications.

Each protein in the trimolecular com-plex interacts with the RNA internal loopin a similar manner as in the bimolecularRNA–protein complex (Fig. 2). The spac-ing of the two RNA internal loops leadsto the positioning of the two U1A pro-teins on one face of the RNA. There areprotein–protein interactions between thetwo U1A monomers that stabilize the tri-molecular complex. A C-terminal α-helix (helix C) mediates theprotein–protein interaction; this helix isstabilized upon formation ofprotein–protein interactions and isamphipathic. The hydrophobic face ofthe helix forms the protein–proteininterface. The structure immediatelyexplains the observed cooperativity ofU1A protein binding to PIE RNA: achange in the spacing of the two bulgeswould twist the two protein monomerswith respect to each other, and pushthem either too close together or too farapart.

The structure provides insights intohow the activity of poly(A) polymerase isregulated by U1A protein’s interactionwith PIE RNA. The trimolecular complexpresents amino acids critical for interac-tion with poly(A) polymerase on one sur-face. Biochemical analyses identified basic

residues adjacent to helix C that arerequired for inhibition of polydenylation9.Formation of the trimolecular complexlikely stabilizes the conformation of theseresidues, and leads to a larger surface areaof interaction with poly(A) polymerase.Because helix C changes its position uponinteraction of U1A with PIE RNA, the cor-rect interaction of U1A with poly(A) poly-merase could only occur in the context ofthe trimolecular complex. The interactionof the two U1A proteins with poly(A)polymerase is reminiscent of dimerizationof transcription factors on DNA, and sub-sequent interaction with the transcriptionmachinery.

The trimolecular complex of two U1Aproteins and PIE RNA (38 kDa RNA–pro-tein complex), is the largest protein–RNAcomplex solved to date. To accomplish thisfeat, Varani and colleagues5 used a barrageof NMR methods to solve the structure(Fig. 2). They simplified the NMR spectraby making a fully symmetric dimer;because each monomer in the dimer hasthe same chemical environment, only oneset of resonances was observed. The spec-tral quality was somewhat reduced in com-parison to the monomeric structure, butthey used partial deuteration to improvelinewidths. Deuteration eliminates path-ways for magnetization transfer, whichcause relaxation of a spectroscopic transi-tion, and subsequent line broadening. Themost difficult regions of protein structureto define by NMR are often dimer inter-faces, because it is difficult to distinguishintrasubunit and intersubunit nuclearOverhauser effects (NOEs, which are thenormal source of distance restraints fortwo protons separated by less than 5–6 Å).They resolved the dimer interface by usingmixtures of isotopically labeled proteins

264 nature structural biology • volume 7 number 4 • april 2000

and unlabeled proteins, and by applyingisotope filter NMR methods10. Finally, theygained some long-range information bycovalent addition of a spin-label, and sub-sequent determination of resonances thatwere paramagnetically broadened; a con-servative limit of 25 Å from the spin labelwas used in structure calculations. Fromthe paramagnetic broadening experiment,the authors obtained much longer-rangedistance information than from just NOEmeasurements, which improved the deter-mination of the global fold of the complex.The final structure was well defined by theNMR data, with a heavy atom root meansquare (r.m.s.) deviation of 2.76 Å for thefinal ensemble of calculated structures; theoverall definition of the trimolecular com-plex is not much worse than that of thebimolecular complex8 previously solved.

The structure presented by Varani andcoworkers5 explains how a specific biologi-cal event is controlled by an RNA–proteininteraction. The specificity of control isbuilt from distinct RNA–protein contacts,which in turn change the conformation ofthe protein, which subsequently interactswith the poly(A) polymerase, controllingits enzymatic activity. The complex net-work of RNA–protein interactions thatcontrol gene expression in eukaryotes willsurely contain other examples of this strat-egy. The current example demonstratesthe powerful combination of NMR struc-tural studies with biochemical analysesthat will provide an avenue for under-standing biological function.

Joseph D. Puglisi is in the Department of Structural Biology, Stanford UniversitySchool of Medicine, Stanford, California94305-5126, USA. email: puglisi@stanford.edu

1. Varani, G. & Nagai, K. Ann. Rev. Biophys. Biomol.Struct. 27, 407–445 (1998).

2. Boelens, W. C. et al. Cell 72, 881–892 (1993).3. Van Gelder, C. W. G. et al. EMBO J. 12, 5191–5200

(1993).4. Gunderson, S. I. et al. Cell 76, 531–541 (1994).5. Varani, L., Gunderson, S. I., Mattaj, I. W., Kay, L.,

Neuhaus, D., Varani, G. Nature Struct. Biol. 7,329–335 (2000).

6. Nagai, K. Oubridge, C., Jessen, T.H., Li, J. & Evans,P.R. Nature 348, 515–520 (1990).

7. Oubridge, C., Ito, N., Evans, P. R. Teo, C.–H. & Nagai,K. Nature 372, 432–438 (1994).

8. Allain, F.H.T. et al. Nature 280, 646–650 (1996).9. Gunderson, S.I., Vagner, S., Polycarpou-Schwartz,

M. & Mattaj, I.W. Genes & Dev. 11, 761–773 (1997).10. Zwahlen, C. et al. J. Am. Chem. Soc 119, 6711–6721

(1997).

Fig. 2 Three-dimensional structure of the tri-molecular complex of two U1A proteins (shownin purple) and PIE RNA. The dimer interface isformed by the interactions of helix C from oneprotein with its partner in the second.

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