REVIEWS
TIBS 25 – MAY 2000
Proteins binding to duplexed
RNA: one motif, multiple
functions
Ivo Fierro-Monti and Michael B. Mathews
Highly structured and double-stranded (ds) RNAs are adaptable and potent
biochemical entities. They interact with dsRNA-binding proteins (RBPs), the
great majority of which contain a sequence called the dsRNA-binding motif
(dsRBM). This ~70-amino-acid sequence motif forms a tertiary structure
that interacts with dsRNA, with partially duplexed RNA and, in some cases,
with RNA–DNA hybrids, generally without obvious RNA sequence specificity. At least nine families of functionally diverse proteins contain one or
more dsRBMs. The motif also participates in complex formation through
protein–protein interactions.
THE CONSERVED CORE of residues
that define the dsRBM (sometimes referred to as the dsRB domain, dsRBD or
DRBD) characterizes a set of proteins
that bind to structured RNA. Among the
first to be recognized were Staufen, responsible for mRNA localization in
Drosophila, and PKR, a dsRNA-activated
protein kinase in mammals1–3. Currently,
the list includes .100 homologous sequences, a selection of which is displayed in Fig. 1. Inspection of the full
dsRBM consensus, shown near the bottom of the alignment, reveals scattered
conservation of amino acids throughout
a sequence extending across 65–75
residues. Conservation is strong over
the C-terminal one-third of the motif and
more widely scattered in its N-terminal
two-thirds. Direct evidence for a role in
RNA binding is presently available only
for a subset of the dsRBMs (denoted 1
in Fig. 1), but the consensus of these sequences closely matches the full consensus and also correlates with important features of the three-dimensional
structure of the dsRBM.
The structures of dsRBMs present in
several proteins from different organisms (asterisked in Fig. 1) have been
I. Fierro-Monti* and M.B. Mathews are at the
Dept of Biochemistry and Molecular Biology,
New Jersey Medical School, UMDNJ, 185 South
Orange Ave., Newark, NJ 07103-2714, USA.
Email: [email protected]
*Present address: Dept of Biochemistry, University
of Cambridge, Cambridge, UK CB2 1QW.
examined by nuclear magnetic resonance (NMR) and X-ray crystallography,
with highly consistent results. As determined by NMR studies of Escherichia
coli RNase III (Ref. 4), Drosophila
melanogaster Staufen5 and the two
dsRBMs of human PKR (Ref. 6), the
motif is characterized by a fold comprising (from the N-terminus): a-helix 1, bstrand 1, b-strand 2, b-strand 3 and ahelix 2 (Figs 1 and 2a). The two a helices
are packed along a face of a threestranded antiparallel b sheet, with most
of the potential RNA-binding residues
exposed on one surface.
RNA binding to the dsRBM
RNA–protein interactions are manifested in the 1.9 Å resolution crystal
structure7 of dsRBM2 from the Xenopus
laevis protein Xlrbpa complexed with
dsRNA, shown in Fig. 2a. Two dsRNAs,
each 10 bp long, are stacked end-wise to
approximate a continuous helix, 16 base
pairs of which are covered by the
dsRBM. The RNA is predominantly Aform, and contacts with the motif take
place on one face of the duplex, spanning two minor grooves and the major
groove between them. Three structural
elements of the dsRBM engage RNA
(Fig. 2a): in regions 1 and 2, the N-terminal a helix (a1) and the loop between b1
and b2 interact with adjacent minor
grooves; in region 3, a2 interacts with
the intervening major groove of dsRNA.
The major groove seems to be wider
than expected for continuous dsRNA,
0968 – 0004/00/$ – See front matter © 2000, Elsevier Science Ltd. All rights reserved.
suggestive of partial unwinding of the
A-form duplex. Consistent with this finding, the dsRBM can bind sequences containing mismatches and non-contiguous
duplex regions3,8,9, potentially allowing for a degree of specificity in
dsRBM–RNA interactions.
The structure in Fig. 2a is compatible
with biochemical and mutagenic analyses, demonstrating the participation of
specific conserved dsRBM residues
(highlighted in Fig. 1) in its interactions
with dsRNA. For instance, the Glu side
chain (E119) in a1 and the His residue
(H141) in region 2 are involved in direct
and water-mediated interactions with
the RNA minor groove. Two Lys
residues (K163 and K167) in a2 interact
with opposing faces of the major
groove, their side chains both buttressed by two aromatic side chains in
b2 and b1 (F145 and Y131, respectively).
Most of these protein–RNA interactions
are with the phosphodiester backbone
or ribose 29-OH groups, which accounts
for the discrimination against DNA. The
limited number of base-specific interactions makes it likely that any specificity
in RNA binding rests on sequence-specific structural features in the duplex. In
keeping with this view, internal loops in
dsRNA appear to play an important role
in defining sites of cleavage by RNase
III and of deamination by ADAR1
(Refs 10,11).
Tertiary structures resembling this
a-b-b-b-a fold are found in other RBPs,
as exemplified in Fig. 2b. These include
ribosomal protein S5 (Ref. 5) and the
polynucleotidyl transferases RNase H,
HIV-1 integrase and MuA transposase7,
although these proteins differ from the
dsRBM family in possessing a b-b-b-a
fold without a1. Some authors have
distinguished a subset of dsRBM
sequences, referred to as type B, which
retain good conservation in a2 (major
groove binding) but diverge from the
consensus elsewhere1,12. Although such
dsRBMs bind dsRNA less well13, they
can still contribute to RNA and protein
binding, as discussed below. However,
not all A-type dsRBM sequences are
equivalent in RNA binding12, and further
structures are needed to disclose the
fine points of their interactions with
RNA.
Nine protein families (at least)
The dsRBM is found in proteins (and
protein sequence fragments) from diverse sources, including viruses, bacteria, and lower and higher eukaryotes.
Considering sequence homology, both
PII: S0968-0004(00)01580-2
241
REVIEWS
TIBS 25 – MAY 2000
*DmSTAU/485-555
CHVP1/202-267
Atbacf21m12/16-82
QRPFPPKFPS-RFALP-PPL--GAHVHHGPNGPFPSVPTPPS-KITL-F-VGKQ------KFVGI-GRTLQQAKHDAAARALQVL
YKDRLLKHTRKVELPR-PEF--VSVFEKGG-----ANPS--F-IVDV-V-INGQK-----ISTGT-GKSRKDAEQNASKIALHTM
FKSRLQEYAQKYKLPT-PVY--EIVK-EGP----SHKSL--F-QSTV-I-LDGVRY----NSLPG-FFNRKAAEQSAAEVALREL
+ CbRNaseIII/154-220
+* EcRNaseIII/156-223
+ StRNaseIII/155-221
+ HiRNaseIII/154-224
+ RcRNaseIII/158-224
+ HpRNaseIII/170-236
+ MtRNaseIII/162-227
+ BsRNaseIII/176-242
+ SpRNaseIII/170-238
+ HsPACT/127-192
+ MmPDRBP/159-224
+ HsTRBP/160-225
+* XlRBPA/113-178
+ SsRNaseIII/161-227
AKSLLQEWLQARRLPL-PTY--EVKI-TGE----AHAQT--F-TVNC-Y-VKGLPH----KTEGV-NTTRRRAEQIAAKRFLELL
PKTRLQEYLQGRHLPL-PTY--LVVQVRGE----AHDQE--F-TIHC-Q-VSGLSE----PVVGT-GSSRRKAEQAAAEQALKKL
PKTRLQEYLQGRHLPL-PSY--LVVQVRGE----AHDQE--F-TIHC-Q-VSGLSE----PVVGT-GSSRRKAEQAAANS-VKKL
AKTRLQEYLQGKHLPL-PTY--EVVNIQGE----AHCQI--F-TVKC-K-VKSAEKI-DRTFVAK-GSSRRKAEQAAAEQILKEL
PKTALQEWAQARGLPP-PRY--ETLGRDGP----DHAPQ--FRIAVV-L-ASGE------TEEAQ-AGSKRNAEQAAAKALLERL
YKTALQELTQAQFCVI-PTY--QLLKEKGP----DHHKE--F-EMAL-Y-IQDKM-----YATAK-GKSKKEAEQQCAYQALQKL
WKTSLQELTAARGLGA-PSY--LVTS-TGP----DHDKE--F-TAVV-V-VMDSE-----YGSGV-GRSKKEAEQKAAAAAWKAL
FKSQLQEYVQRDGKGS-LEY--KISNEKGP----AHNRE--F-EAIV-S-LKGEP-----LGVGN-GRSKKEAEQHAAQEALAKL
YKTELQEFLQAGDART-LEY--KLIKESQP---LEGNRV--L-YTVV-AEIGGIR-----YGEGC-GYTHKEAEQLAARDALQKL
PIGSLQELAIHHGWRL-PEY--TLSQEGGP----AHKRE--Y-TTIC-R-LESF------METGK-GASKKQAKRNAAEKFLAKF
PVGALQELVVQKGWRL-PEY--MVTQESGP----AHRKE--F-TMTC-R-VERF------IEIGS-GTSKKLAKRNAAAKMLLRV
PVGALQELVVQKGWRL-PEY--TVTQESGP----AHRKE--F-TMTC-R-VERF------IEIGS-GTSKKLAKRNAAAKMLLRV
PVGSLQELAVQKGWRL-PEY--TVAQESGP----PHKRE--F-TITC-R-VETF------VETGS-GTSKQVAKRVAAEKLLTKF
VKSMLQQWALAKTKQL-PEY--ELINTSGP----PHAQE--F-TFTV-K-VAGKI-----HGQGS-GPSKQIATKQAALEALKSL
RnRED/127-190
HsRED/ 79-142
RnRED/ 79-142
HsNF90/403-466
Xl4f1/403-466
Xl4f2/305-368
MnSPNR/388-451
RnRED/285-346
HsRED/236-296
RnRED/236-296
HsNF90/530-593
MnSPNR/511-574
Xl4f1/522-585
Xl4f2/424-487
Cef39e9.7/35-100
Cef55a4/129-194
CeSTAU/414-479
−* DmSTAU/712-779
PKNALVQLHE--LKPG-LQY--RMVSQTGP----VHAPV--F-AVAV-E-VNGL------TFEGT-GPTKKKAKMRAAEMALKSF
PKNALMQLNE--IKPG-LQY--TLLSQTGP----VHAPL--F-VMSV-E-VNGQ------VFEGS-GPTKKKAKLHAAEKALRSF
PKNALMQLNE--IKPG-LQY--MLLSQTGP----VHAPL--F-VMSV-E-VNGQ------VFEGS-GPTKKKAKLHAAEKALRSF
AMNALMRLNQ--LKPG-LQY--KLVSQTGP----VHAPI--F-TMSV-E-VDGN------SFEAS-GPSKKTAKLHVAVKVLQDM
VMNALMRLNQ--LKPG-LQY--KLISQTGP----VHAPV--F-TMSV-E-VDDK------TFEAS-GPSKKTAKLHVAVKVLQDM
AMNALMRLNQ--LKPG-LQY--KLISQTGP----VHAPI--F-TMSV-E-VDDK------TFEAS-GPSKKTAKLHVAVKVLQDM
LMNALMRLNQ--IRPG-LQY--KLLSQSGP----VHAPV--F-TMSV-D-VDGT------TYEAS-GPSKKTAKLHVAVKVLQAM
PVVVLNELRS--GLRY-VCL--SETA-EKP-----RVKS--F-VMAV-C-VDGR------TFEGS-GRSKKLAKGQAAQAALQAL
PVMILNELRP-----G-LKY--DFLSESGE----SHAKS--F-VMSV-V-VDGQ------FFEGS-GRNKKLAKARAAQSALAAI
PVMILNELRP-----G-LKY--DFLSESGE----SHAKS--F-VMSV-V-VDGQ------FFEGS-GRNKKLAKARAAQSALATV
GKNPVMELNEKRRGL---KY--ELISETGG----SHDKR--F-VMEV-E-VDGQ------KFQGA-GSNKKVAKAYAALAALEKL
GKNPVMELNEKRRGL---KY--ELISETGG----SHDKR--F-VMEV-E-VDGQ------KFRGA-GPNKKVAKASAALAALEKL
GKNPVMELNEKRRGL---KY--EMISETGG----SHDKR--F-VMEV-E-VDGV------KFQGS-GSNKKVAKAYAALSALEKL
GKNPVMELNEKRRGL---KY--ELISETGG----SHDKR--F-IMEV-E-VDGV------KFQGN-GSNKKVAKAYAALSALEKL
PVAQLNEYAQKFYKKY-PTF--EFVKEQAV----GKHRV--F-IIQA-T-FEDK------TLEGR-GPSKMIAKRAAAEAILESI
PVAQLNEYAQKFYKKY-PTF--EFVKEQAV------GKHREF-VIQA-T-FENK------TLEGR-GPSKIISKRAAAEAILESI
PVSRLIQVTQAKSKEH-PTF--ELVAEHGV----SKYKE--F-IIQV-K-YGDD------VQEGK-GPNKRLAKRAAAEAMLESI
PITKLIQLQQTRKEKE-PIF--ELIAKNGN----ETARRREF-VMEV-S-ASGS------TARGT-GNSKKLAKRNAAQALFELL
− HsPACT/35-99
− MmP1RBP/31-95
− HsTRBP/31-95
−* XlRBPA/21-85
+* DmSTAU/579-643
−* DmSTAU/312-376
CeSTAU/79-144
Cef55a4/29-99
CeSTAU/293-363
PIQVLHEYGM-KTKNI-PVY--ECER-SDV---QIHVPT--F-TFRV-T-VGDI------TCTGE-GTSKKLAKHRAAEAAINIL
PISLLQEYGT-RIGKT-PVY--DLLKEEGQ----AHQPN--F-TFRV-T-VGDT------SCTGT-GPSKKAAKHKAAEVALKHL
PISLLQEYGT-RIGKT-PVY--DLLKAEGQ----AHQPN--F-TFRV-T-VGDT------SCTGQ-GPSKKAAKHKAAEVALKHL
PIQLLHEFGT-KTGNH-PVY--TLEKAEGQ----AHNPS--F-TFRL-V-IGDI------TSLGE-GPSKKTPKQKAAEFALNIL
PISQVHEIGI-KRNMT-VHF--KVLREEGP----AHMKN--F-ITAC-I-VGSI------VTEGE-GNGKKVSKKRAAEKMLVEL
PMCLVNELAR-YNKIT-HQY--RLTEERGP----AHCKT--F-TVTL-M-LGDE------EYSAD-GFKIKKAQHLAASKAIEET
AMCRVAEIAR-FNKLR-HVY--NLQDESGP----AHKKL--F-TVKL-V-LTEAE-----TFEGS-GTSIKRAQQASAEAALKGT
VISIIHEKAQ-QLKLK-INF--EVLEEGGD----QHNRQYAV-RYTL-V-ADDNVVK--AKAMGK-GKNKKSAQQEACTQLLATV
VISDIHEKAY-QLKVN-VVF--EVLKEEGP----PHDRQ--Y-VVRCAF-VTSGNVV-KAEAVGK-GKKKKSAQQEACTQLLATV
HsADAR/504-569
RnADAR/454-519
— XlADAR/575-640
— HsADAR/727-792
RnADAR/673-738
— XlADAR/797-862
HsADAR/615-680
RnADAR/565-630
— XlADAR/680-745
+* HsPKR/10-75
+ MmPKR/9-74
+ RnPKR/9-74
+ VARVE3/120-184
+ VACVE3/118-182
+ VACVE3/118-182
PISGLLEYAQ-FASQT-CEF--NMIEQSGP----PHEPR--F-KFQV-V-INGRE-----FPPAE-AGSKKVAKQDAAMKAMTIL
PVSGLLEYAQ-FTSQT-CDF--NLIEQSGP----SHEPR--F-KFQV-V-INGRE-----FPPAE-AGSKKVAKQDAAVKAMAIL
PVSGLLEFTH-YCSQQ-CDF--ALLNQSGP----SHDPR--F-KIQA-V-IDGRR-----FPVAE-ANSKKTAKKDAAALALRIL
PVGGLLEYAR-SHGFA-AEF--KLVDQSGP----PHEPK--F-VYQA-K-VGGRW-----FPAVC-AHSKKQGKQEAADAALRVL
PVGGLLEYAR-SHGFA-AEF--KLIDQSGP----PHEPK--F-VYQA-K-VGGRW-----FPAVC-AHSKKQGKQDAADAALRVL
PVSGLLEYAR-AKGFA-AEF--KMVNQTGP----PHDPK--F-VFQA-K-VGGRW-----FPAVS-ASNKKQAKAEAADAALRVL
PVTTLLECMH-KLGNS-CEF--RLLSKEGP----AHEPK--F-QYCV-A-VGAQT-----FPSVS-APSKKVAKQMAAEEAMKAL
PVTTLLECMH-KLGNS-CEF--RLLSKEGP----AHDPK--F-QYCV-A-VGAQT-----FPSVS-APSKKVAKQMAAEEAMKAL
PISLLMEHGQ-KSGNM-CEF--QLVSQEGP----PHDPK--F-TYTV-K-IGNQT-----FPPVV-ANNKKMAKHLAAEAAVREL
FMEELNTYRQ-KQGVV-LKY--QELPNSGP----PHDRR--F-TFQV-I-IDGRE-----FPEGE-GRSKKEAKNAAAKLAVEIL
YMDKLNKYRQ-MHGVA-ITY--KELSTSGP----PHDRR--F-TFQV-L-IDEKE-----FPEAK-GRSKQEARNAAAKLAVDIL
YVDKLNKYSQ-IHKVK-IIY--KEISVTGP----PHDRR--F-TFQV-I-IEERE-----FPEGE-GRSKQEAKNNAAKLAVEIL
PVTIINEYCQ-ITKRD-WSF--RIES-VGP----SNSPT--F-YACV-D-IDGRV-----FDKAD-GKSKRDAKNNAAKLAVDKL
PVTIINEYCQ-ITKRD-WSF--RIES-VGP----SNSPT--F-YACV-D-IDGRV-----FDKAD-GKSKRDAKNNAAKLAVDKL
PVTVINEYCQ-ITRRD-WSF--RIES-VGP----SNSPT--F-YACV-D-IDGRV-----FDKAD-GKSKRDAKNNAAKLAVDKL
DmRHA/3-68
HsRHA/4-69
BtRHA/4-69
CeRHA/106-172
DmRHA/170-239
HsRHA/181-250
BtRHA/178-247
CeRHA/273-343
IKSFLYQFCA-KSQIE-PKF--DIRQ-TGP----KNRQR--F-LCEV-R-VEPNTY----IGVGN-STNKKDAEKNACRDFVNYL
VKNFLYAWCG-KRKMT-PSY--EIRA-VGN----KNRQK--F-MCEV-Q-VEGYNY----TGMGN-STNKKDAQSNAARDFVNYL
VKNFLYAWCG-KRKMT-PSY--EIRA-VGN----KNRQK--F-MCEV-R-VEGYNY----TGMGN-STNKKDAQSNAARDFVNYL
VKEFLYAWLGKNKYGN-PTY--DTKS-ETR----SGRQR--F-KCEL-R-ITGFGY----TAFGN-STNKKDAATNAAQDFCQYL
AKERLNIYKQ-TNNIR-DDY--KYTP-VGP----EHARS--F-LAEL-S-IYVPALNRTVTARES-GSNKKSASKSCALSLVRQL
AKARLNQYFQ-KEKIQ-GEY--KYTQ-VGP----DHNRS--F-IAEM-T-IYIKQLGRRIFAREH-GSNKKLAAQSCALSLVRQL
AKARLNQYFQ-KEKIQ-GEY--KYTQ-VGP----DHNRS--F-IAEM-T-IYIKQIGRRIFAREH-GSNKKLAAQSCALSLVRQL
SKKALNEFLQKMRLPQ-VNYGTKIRESNTV----KTMET----TAQI-F-VPQINKN--LVGKGT-GSNKKVSEAACAMNIVRQM
−* HsPKR/101-165
− MmPKR/96-160
− RnPKR/96-160
YIGLINRIAQ-KKRLT-VNY--EQCA-SGV----HGPEG--F-HYKC-K-MGQKE-----YSIGT-GSTKQEAKQLAAKLAYLQI
YIGLVNSFAQ-KKKLS-VNY--EQCE-PNS----ELPQR--F-ICKC-K-IGQTM-----YGTGS-GVTKQEAKQLAAKEAYQKL
YIGLVNSFAQ-KENLP-VNF--ELCD-PDS----QLPHR--F-ICKC-K-IGQTT-----YGTGF-GANKKEAKQLAAKNAYQKL
Cet20g5.11/170-232
Atbacf21m12/102-168
HsSON/1365-1432
ScMRL3P/303-370
CeHelYM68/1746-1806
MmTNRBP/139-204
*DmSTAU/951-1016
Full consensus (>50%)
(+) (>70%)
WVGKLQEKSQKSKLQA-PIY--EDSK---N----ERTER--F-LVIC-T-MCNQ------KTRGI-RSKKKDAKNLAAWLMWKAL
CKNLLQEYAQ-KMNYA-IPL--YQCQKVET----LGRVT-QF-TCTV-E-IGGIK-----YTGAA-TRTKKDAEISAGRTALLAI
PVSALMEICNKRRWQP-PEF--LLVHDSGP----DHRKH--F-LFRV-L-INGSAY----QPSFA-SPNKKHAKATAATVVLQAM
PTRELAMLCR-REGLEKPVS--KLVAESGR-----LSKSPVF-IVHV-F-SGEET-----LGEGY-GSSLKEAKARAATDALMKW
PIRELMEFEQSKVRFS-KME--RILE-SGK------VRV----TVEV-V-NNMR-------FTGM-GRNYRIAKATAAKRALKYL
PVSALHQFAQ-MQRVQ-LDL--KETVTTGN----VMGPY--F-AFCA-V-VDGIQ-----YKTGL-GQNKKESRSNAAKLALDEL
HMKEQLLYLSKLLDFE-VNF--SDYP-KGN-----HNEF--L-TIVT-L-STHPPQ----ICHGV-GKSSEESQNDAASNALKIL
P---L-E--Q---------Y--------GP-----H-----F----V-----G----------G--G-SKK-AK--AA--AL--L
P---L-E------------Y--------GP-----H-----F------------------------G-SK--A---AA------L
α1
Region 1
β2
β1
Region 2
β3
α2
Region 3
Ti BS
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within the motifs and outside them, as
well as the number and distribution of
their dsRBMs, most of the proteins fall
into one of nine major groups (see Fig. 1
and Table 1). Prototypical representatives of these nine dsRBP families are
drawn schematically in Fig. 3, which diagrams the patterns of dsRBMs and additional conserved domains that characterize some of the families. These
additional domains are associated with
the proteins’ individual functions and, in
several cases, have been implicated in
their catalytic activities, notably the helicase activity of RHA (Ref. 14), adenosine deaminase activity of ADAR1
(Ref. 15) and ADAR2 (Ref. 16), protein
kinase activity of PKR (Ref. 17) and
ribonuclease activity of RNase III
(Ref. 10). In many (but not all) proteins,
the dsRBMs are also proven to be
critical for function.
Strikingly, most dsRBP families contain multiple dsRBMs. Four families contain two dsRBMs, whereas ADAR1 has
three and Staufen boasts as many as five
(Drosophila) or four (human) copies of
the motif. Members of the TRBP family
have either two or three copies. The
RNase III and E3L families are exceptional, making do with only one (although RNase III probably functions as a
dimer). It is clear from studies of several
proteins that dsRBMs vary in their affinity for dsRNA. In PKR, for example,
dsRBM1 (type A; nearer the N terminus)
binds much more effectively than
dsRBM2 (type B), but both are needed
for optimal binding13,18. This cooperativity suggests that multiple dsRBMs might
facilitate the interaction of a protein
with specific target RNAs. The spacing
of the motifs within a protein might also
be related to its binding specificity and
function. Some dsRBMs have been
shown to play a role in protein dimerization19–21, so another possibility is that
multiple dsRBMs represent a feature
that stabilizes homo- or heterotypic protein–protein interactions. Such interactions often subserve a regulatory function, as described below, but in many
cases it remains to be established
(a)
β3
α2
β2
Loop between
β1 and β2
β1
α1
(b)
Xlrbpa2–dsRNA
C
RNase H
HIV-1 integrase
MuA transposase
N
Bound
sulfate
Bound
metals
Ti BS
Figure 2
Double-stranded RNA-binding motif (dsRBM) and related structures. (a) Structure of the
complex between Xlrbpa dsRBM2 and dsRNA (Ref. 7). Side chains shown in green and gray
represent residues from regions 1 and 2 that interact with the RNA minor groove and major
groove, respectively. Hydrogen bonds are represented by dotted black lines. Oxygen, nitrogen and carbon atoms are shown in red, blue and yellow, respectively. Residue numbers
correspond to the full-length Xlrbpa protein. (b) Similar folds are found in polynucleotidyl
transferases. The b-b-b-a folds of Xlrbpa dsRBM2, RNaseH, HIV-1 integrase and MuA transposase are shown in black. In all cases, the loop between the N-terminal part of the C-terminal a helix and b strands 1 and 2 is proposed to interact with nucleic acids. Reproduced,
with permission, from Ref. 7.
whether the interaction is direct or
bridged by dsRNA or other components.
Are there families of dsRBPs beyond
the nine depicted in Fig. 3? Some dsRBM
sequences (shown in black and white in
Fig. 1) do not seem to match any of
the nine families of dsRBM-containing
proteins. Whether these sequences
belong to dsRBPs has not yet been
experimentally determined, but it is
likely that at least some of them represent dsRBM-containing protein families
whose natures and functions are
presently unknown. In addition, there are
Figure 1
Double-stranded RNA-binding motif (dsRBM) sequences. The panel displays a multiple alignment of 80 dsRBM sequences clustered into protein sequence families using the PFAM domain alignment database51. Residues that are .50%, .70% and .80% conserved are shown in
light blue, dark blue and red, respectively. The full consensus is defined as residues that are .50% conserved among all dsRBM sequences;
the (1) consensus consists of residues that are .70% conserved among those sequences that are known to bind dsRNA strongly. A
schematic representation of the dsRBM sequence fold, its component structural elements and the three regions involved in RNA interactions
(see Fig. 2) are shown at the bottom of the alignment. Conserved residues involved in RNA interactions and discussed in the text are highlighted in yellow. Along the left side, dsRBM sequences are named according to the species of origin, protein name and residue numbers.
dsRBM sequences present in nine protein families (see text, Table 1 and Fig. 3) are grouped in blocks highlighted in various colors; more distantly related dsRBM sequences, including some that could represent founder members of new families, are not colored. (1) indicates that
the dsRBM is functional in dsRNA binding; (2) signifies weak or undetectable binding; dsRBMs not tested for dsRNA binding lack (1) or (2).
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Table 1. Functions of double-stranded RNA-binding proteins
dsRBP
Enzymatic function
Other functions and characteristics
PKR
Ser/Thr kinase
Phosphorylation of eIF2a
IFN-induced antiviral response
Translation, transcription, transformation, apoptosis
TRBP
PKR activator (PACT)
PKR inhibitor (TRBP)
Ribosomal and hnRNA association (Xlrbpa)
E3L
RHA
Viral PKR inhibitor
RNA/DNA helicase
NF90
ADAR1
ADAR2
NFAT transcription factor, PKR activator
Mitosis phosphorylation (MPP4)
Translational activator? RNA transport? (Spnr)
A→I deaminase
Staufen
RNase III
Transcriptional coactivator (RHA)
Dosage compensation (MLE)
RNA transport?
Site-specific pre-mRNA editing
IFN-induced
mRNA localization
Translational activation?
dsRNA-specific endoribonuclease
Processing of rRNA and other precursors
Abbreviations: ADAR, dsRNA-specific adenosine deaminase; dsRBP, double-stranded RNA-binding
protein; NF90, nuclear factor 90; PKR, dsRNA-dependent protein kinase; RHA, RNA helicase A; TRBP,
TAR RNA-binding protein.
a few proteins that bind dsRNA without
having a dsRBM. Non-dsRBM-containing
dsRBPs include the influenza virus nonstructural protein NS1 and 29,59-oligoadenylate synthetase (which, nevertheless,
shares the features of interferon
inducibility and dsRNA activation with
PKR).
Functions of dsRNA-binding proteins
The nine families of dsRBPs in Fig. 3
can be divided into two groups: enzymes with well-established activities
and RBPs to which no enzymatic function has (yet) been attributed. The enzyme group includes RHA, RNase III,
ADAR1 and ADAR2, all of which modify
RNA structure (via unwinding, cleavage
and deamination), and PKR, for which
dsRNA is an effector. The second group
includes Staufen and E3L, whose functions entail RNA binding, and two protein families (NF90 and TRBP) for which
the significance of RNA binding is enigmatic. Table 1 summarizes the principal
features of these protein families, and
thumbnail sketches of their properties
and functions follow.
PKR. The RNA-dependent protein kinase PKR is the most intensively studied
dsRBP. Its synthesis is induced by the
anti-viral cytokine interferon, and its activation by dsRNA (and some other polyanions) is accompanied by autophosphorylation17. Activation also requires PKR
dimerization, which is mediated by its
dsRBMs20 and other sequences. The
dsRBMs of PKR resemble a molecular
244
switch that regulates its kinase activity:
dsRNA binding appears to trigger a conformational change that unmasks the enzyme’s catalytic domain22. Activated PKR
phosphorylates the a subunit of eukaryotic translation initiation factor 2 (eIF2a),
thereby inhibiting protein synthesis in response to viral infection and other stimuli. The growing list of additional, albeit
less well-established, PKR substrates includes ikB, the inhibitory subunit of
transcription factor NFkB, the HIV-1 transcriptional activator Tat, the tumor suppressor p53, and the dsRBPs NF90 and
RHA. PKR is implicated in several key cellular processes17,23, of which the anti-viral
response is best understood. Many
viruses have developed strategies to
cope with the threat to their multiplication posed by PKR activation24: for example, the highly structured adenovirus
VA RNAI binds to the dsRBMs without activating the enzyme. A few cellular mRNAs
have also been implicated in PKR regulation as inhibitors25 or as activators9,26,27.
TRBP. The transactivation region
(TAR)-RNA-binding protein, TRBP, is a
human homologue of the X. laevis protein Xlrbpa. It is able to bind to a
number of structured RNAs, including
dsRNA, and, like PKR, it forms homodimers. TRBP heterodimerizes with PKR
(Ref. 19), which could explain its inhibitory effect on PKR function and its
ability to transform 3T3 cells28. Another
member of the TRBP family is the cellular protein PACT that, in contrast, activates PKR (Ref. 29). Xlrbpa associates
with ribosomes and heterogeneous
nuclear RNA (Ref. 30). No additional
functional domains or enzymatic activities
have been associated with this family of
proteins to date, and their biological
roles remain to be elucidated.
E3L. The E3L protein found in
poxviruses, exemplified by vaccinia
virus, is another dsRBP that can heterodimerize with and inhibit PKR
(Ref. 21). This interaction represents
one means whereby poxviruses neutralize the PKR-mediated cellular antiviral
response (another being competition by
the viral K3L protein, which acts as a
pseudosubstrate). E3L’s mode of action
is surprisingly complex. It binds to PKR
through its N-terminal region as well as
its C-terminal dsRBM, via both protein–protein and RNA-mediated dsRBM
associations21. Interactions between
dsRBPs are likely to be facilitated by
RNA bridging through their dsRBMs: by
drawing proteins into closer proximity
in this way, RNAs can serve as regulators of several cellular processes.
Conceivably, RNA molecules containing
ds regions could nucleate the assembly
of various dsRBPs engaged in metabolism of structured RNAs.
RHA. Despite its name, RNA helicase
A (RHA) displays both RNA and DNA helicase activity14. Knockout experiments
in mice show that RHA is essential for
gastrulation, specifically for embryonic
mesoderm induction31. It contains two
dsRBMs (Fig. 3), both of which are
necessary for efficient binding to dsRNA.
However, their deletion does not abolish
the enzyme’s unwinding activity32. Many
lines of evidence point to a role in transcription. The Drosophila homologue of
RHA, the maleless protein, is required
for upregulating X chromosome transcription in the early stages of male fly
development (dosage compensation)14.
In mammals, RHA interacts with RNA
polymerase II and the transcriptional
coactivator CBP/p300 to stimulate transcription33. It has been proposed that
RHA melts DNA–RNA hybrids, such as
those that occur during transcription32.
A role for RHA in post-transcriptional
processes has also been inferred.
RHA shuttles between the nucleus and
cytoplasm, and interacts with the
constitutive transport element (CTE)
of simian retrovirus34. Taken together
with its involvement in HIV Revmediated gene expression35, these data
imply participation in mRNA nuclear
export, which could account for
the presence of dsRBMs in the RHA
family.
TIBS 25 – MAY 2000
REVIEWS
NF90. Nuclear factor 90 (NF90),
Staufen. D. melanogaster Staufen
the prototype for this family, is one of
is a well-studied protein involved in
RHA
Hs
the most abundant dsRBPs in human
localizing maternal mRNAs in the
cells36. Several NF90 homologues
Drosophila oocyte and embryo.
have been isolated, including human
This dsRBP interacts specifically
ADAR1 Hs
MPP4 (Ref. 37), murine ILF3 and Spnr
through one or more dsRBMs with
(Refs 38,39), and two Xenopus prothe 39UTRs of bicoid and oskar
teins 4F.1 and 4F.2 (Ref. 40). NF90 parmRNAs at the anterior pole of the
Staufen Dm
ticipates in numerous interactions
embryo and the posterior pole of
with other macromolecules. It was
the oocyte, respectively46. It also
initially purified, together with
serves to localize prospero mRNA in
ADAR2 Hs
nuclear factor 45 (NF45), by its
neuroblasts properly. Mammalian
ability to bind to a DNA promoter
Staufen homologues were recently
element (ARRE) in the IL-2 gene,
cloned47,48: the mouse and human
and depletion experiments sugproteins are similar to their
Hs
NF90
gested that it functions as a tranDrosophila counterpart, but they
scriptional activator41. NF90 copulack the first dsRBM found in the
rifies with a number of proteins,
Drosophila prototype and contain a
PKR
Hs
including NF45, the DNA-dependent
putative microtubule-binding doprotein kinase (DNA-PK), the DNAmain not found in the Drosophila
binding Ku proteins and eIF2
protein. Human Staufen can interTRBP
Hs
(Ref. 42). Furthermore, NF90 coact with dsRNA and tubulin, and
immunoprecipitates with RHA and
was shown to translocate in a bican activate PKR in yeast (I. Fierrodirectional microtubule-dependent
RNase III Ec
Monti and M.B. Mathews, unpubmanner in rat hippocampal neulished). NF90 also binds to dsRNA
rons49. Staufen associates with the
and VA RNAII, a second, small, highly
rough endoplasmic reticulum and
E3L
Vv
structured adenovirus transcript36.
with polysomes, suggesting a
Ti BS
The functional significance of this
role in connection with the transextensive network of interactions is
lation machinery. Thus, in both
Figure 3
not yet clear, but it is likely that
Drosophila and mammals, Staufen
The nine double-stranded RNA-binding protein (dsRBP)
NF90 participates in DNA and RNA
appears to be involved in targeting
families. Sequence similarities and functional prometabolism or information transfer.
specific mRNAs to intracellular loci
perties define the nine families of double-stranded
ADAR1. The dsRNA-specific
so as to promote their appropriate
RNA-binding motif (dsRBM)-containing proteins. The
dsRBMs are shown in pink, and other conserved
adenosine deaminase family (alias
subcellular distribution.
sequences are in different colors. The families are
dsRAD or DRADA) is involved
RNase III. A numerous family of
named after well-studied prototypes: RHA, RNA heliin the covalent modification of
dsRBPs is represented by the
case A; ADAR, dsRNA-specific adenosine deaminase;
dsRNA (Refs 15,43). It converts
dsRNA-specific endoribonuclease
ADAR2 (or RED), specific RNA editase; NF90, nuclear
many of the adenosine (A)
RNase III of E. coli, which has the
factor 90; PKR, protein kinase regulated by RNA;
residues in duplex RNA to inosine
distinction of being the first dsRBP
TRBP, transactivation region (TAR)-RNA-binding protein;
RNase III, ribonuclease III; E3L, vaccinia virus E3L
(I), leading to a modified product
to be characterized. Members of
gene product. The diagrams refer specifically to
prone to strand separation, and
this family are found in prokaryproteins from the sources indicated (Dm; Drosophila
can cause mRNA recoding when
otes and eukaryotes, although not
melanogaster; Ec, Escherichia coli; Hs, Homo
as yet in higher eukaryotes. Their
substitution of I (read as G) for spesapiens; Vv, vaccinia virus); other family members
best-known function relates to the
cific A residues changes a codon.
retain similar modular structures. Note that the third
Natural ADAR1 substrates include
processing of precursor RNAs, indsRBM of Xlrbpa is not well conserved in TRBP and is
hepatitis-d virus RNA and serocluding precursors to ribosomal,
not recognized in the PFAM alignment.
tonin-2C receptor and glutamine
messenger and small nuclear
receptor (GluR) mRNAs of mamRNAs. Perfect duplexes exceeding
mals43–45. Like PKR, ADAR1 is induced by A→I deamination of pre-mRNA. Its speci- 20 base pairs (i.e. two helical turns of an
interferon, possibly as part of the cellu- ficity differs from that of ADAR1. In GluR-B A-form RNA helix) are recognized as sublar antiviral strategy. The relative impor- mRNA, for example, ADAR1 efficiently strates and are cleaved50, even though
tance of the three dsRBMs for RNA bind- deaminates A residues at an intronic site cleavage sites in natural substrates often
ing and deamination depends in part on and an exonic site, the R/G site, where an consist of internal loops within a region
the nature of the substrate and the en- Arg codon is replaced by a Gly codon. of dsRNA (Ref. 10). The resultant distorzyme isoform; the identity of the en- ADAR2, on the other hand, efficiently tar- tion of the helix might contribute
zyme(s) responsible for individual edit- gets the R/G site and a Q/R site (Gln→Arg), to dsRBM-binding and cleavage-site
ing events remains to be determined.
but attacks the exonic site only weakly43. specificity.
ADAR2. The dsRNA-specific editase Such editing events could be crucial for
(RED) family, now renamed ADAR2 proper gene expression, as is the case for Conclusions and conundrums
(Ref. 43), shares ~31% overall identity with the Q/R reducement in GluR-B, which is
The dsRBM is a widespread structural
ADAR1. The homology is largely within essential for receptor function. However, module subserving a variety of functhe catalytic region (boxed in Fig. 3) and the basis for the high degree of specificity tional roles in at least nine families of
dsRBMs. ADAR2 also causes site-specific of the deaminases is not understood.
highly conserved proteins. It could be
245
REVIEWS
responsible for specific RNA recognition
(as in Staufen) or essential for catalytic
activity (as in ADAR); it can regulate the
functional status of the dsRBP that contains it (as in PKR) or of another dsRBP
(as in E3L); and its presence in proteins
whose functions remain to be defined
suggests that it also plays other roles. A
remarkable feature of the dsRBM is its
ability to mediate both RNA–protein and
protein–protein interactions with other
members of the dsRBP family. This
versatility could be exploited as a
molecular mechanism for bringing RBPs
into proximity through RNA bridging.
Conversely, complexes containing two
or more dsRBPs might shepherd transcripts through various transcriptional
and post-transcriptional events regulating gene expression. In essence, the
dsRBM provides a link between RNAs
and multifunctional proteins and complexes, including those forming part of
the cellular architecture.
Whereas great strides have been
taken in the few years since the motif
was first noted, many issues still remain
to be addressed. For example, what are
the concerted regulatory mechanisms
in which dsRBPs participate? Do specific RNAs target particular dsRBPs and
exert control over their functions? For
those dsRBP–RNA interactions that are
sequence-specific, wherein does their
specificity reside? Is it conferred by protein sequence, within the dsRBM or elsewhere in the RBP, or by complex formation with other proteins? Are there
features of RNA structure that display
variable degrees of affinity for dsRBMs?
Can this property be regulated by physiological conditions? The answers to
such questions will have a broad impact
on biomedical science.
TIBS 25 – MAY 2000
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15
16
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18
19
20
21
Acknowledgements
Our work is supported by grant number AI34552 from the National Institute
of Allergy and Infectious Disease, NIH.
We thank Tsafi Pe’ery for suggestions
and apologize to readers and authors
alike for the omission of many references owing to space constraints.
22
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24
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