(Received for publication, October 27, 1995)
From the
TIA-1 and TIAR are two closely related RNA recognition motif
(RRM) proteins which possess three RRM-type RNA binding domains (RRMs
1, 2, and 3). Although both proteins have been implicated as effectors
of apoptotic cell death, the specific functions of TIA-1 and TIAR are
not known. We have performed in vitro selection/amplification
from pools of random RNA sequences to identify RNAs to which TIA-1 and
TIAR bind with high affinity. Both proteins selected RNAs containing
one or several short stretches of uridylate residues suggesting that
the two proteins have similar RNA binding specificities. Replacement of
the uridylate stretch with an equal number of cytidine residues
eliminates the protein-RNA interaction. Mutational analysis indicates
that, for both TIA-1 and TIAR, it is the second RNA binding domain (RRM
2) which mediates the specific binding to uridylate-rich RNAs. Although
RRM 2 is both necessary and sufficient for this interaction, the
affinity for the selected RNA (as determined by filter binding assays)
does increase when the second domain of TIAR is expressed together with
the first and third domains (K = 2
10
M) rather than alone (K
= 5
10
M). Although RRM 3 (of either TIA-1 or TIAR) does not
interact with the uridylate-rich sequences selected by the full-length
proteins, it is a bona fide RNA binding domain capable of
affinity-precipitating a population of cellular RNAs ranging in size
from 0.5 to 5 kilobases. In contrast, RRM 1 does not
affinity-precipitate cellular RNA. The inability of RRM 1 to interact
with RNA may be due to the presence of negatively charged amino acids
within the RNP 1 octamer.
RNA-binding proteins are involved in a variety of fundamental
cellular processes including RNA splicing, polyadenylation, RNA
transport, and translation. Specific RNA sequences with which these
proteins interact have been identified in some cases, but for the
majority of RNA-binding proteins, the RNA targets are unknown. TIA-1
and TIAR are two closely related members of the RNA recognition motif
(RRM) ()family of RNA-binding
proteins(1, 2) . The RRM (also known as the RNP motif,
the RNP consensus sequence, the RNP-80, and the consensus sequence
RNA-binding domain) consists of 80-90 amino acids containing two
stretches of 8 and 6 highly conserved residues called RNP 1 and RNP 2,
respectively(3, 4, 5) . TIA-1 and TIAR both
possess three amino-terminal RRM domains and a glutamine-rich carboxyl
terminus. The RRM domains of TIA-1 and TIAR are very similar with 79%
amino acid identity between the first domains, 89% amino acid identity
between the second domains, and 91% amino acid identity between the
third domains. The carboxyl termini of the two proteins, in contrast,
are only 51% identical in amino acid sequence(2) .
Several observations suggest that TIA-1 and TIAR are involved in signaling apoptotic cell death. The introduction of purified TIA-1 or TIAR into the cytoplasm of thymocytes permeabilized with digitonin results in fragmentation of genomic DNA into nucleosome-sized oligomers (1, 2) . TIAR is translocated from the nucleus to the cytoplasm in response to exogenous triggers of apoptosis(6) , and TIA-1 is phosphorylated by a serine/threonine kinase that is activated during Fas-mediated apoptosis(7) .
Although these findings implicate TIA-1 and TIAR in the apoptotic process, the specific functions of these proteins have not been determined. The existence of Drosophila(8, 9) and Caenorhabditis elegans(10) homologs, each of which shares 46% amino acid identity with human TIA-1 and TIAR, indicates that TIA-1 and TIAR are proteins which have been evolutionarily conserved. In order to understand the functions of these proteins, both in cells undergoing apoptosis and in healthy cells, it is necessary to identify RNAs with which they interact. As a first step toward identifying their RNA targets, we have determined the RNA binding specificities of both TIA-1 and TIAR by selection/amplification from pools of random RNA sequences(11) . We have found that both of these proteins interact with RNAs containing short stretches of uridylates, and that for both proteins it is the second RNA-binding domain which mediates this sequence specificity.
Truncated and truncated/mutated selected RNA sequences were in vitro transcribed as described above using DNA templates encoding either the first 50 nucleotides of a selected RNA sequence (``truncated selected sequence'') or the first 50 nucleotides of the selected RNA sequence with the uridylate residues of the uridylate stretch replaced by cytidines (``truncated/mutated selected sequence''). The DNA templates were constructed by cloning annealed complementary oligonucleotides encoding the RNA sequences into the EcoRI and BamHI sites of the pSP65 vector.
Figure 1: The sequences of RNAs selected by TIA-1 and TIAR. Selection/amplification from pools of random RNA sequences was carried out as described under ``Experimental Procedures.'' The sequences of 20 TIA-1-selected RNAs and 20 TIAR-selected RNAs are shown. The uridylate stretches are underlined.
Figure 2:
UV-cross-linking of TIA-1 and TIAR to the
selected RNA sequences. A, purified GST-TIA-1 or GST-TIAR was
incubated with P-labeled RNA and UV-irradiated as
described under ``Experimental Procedures.'' Following RNase
A treatment, cross-linked protein was visualized by autoradiography of
10% SDS-polyacrylamide gels. Four TIA-1-selected RNAs (lanes
1-4), four TIAR-selected RNAs (lanes 9-12),
three randomly picked RNAs (lanes 5-7 and 13-15), or a mixture of 20 randomly picked RNAs (lanes 8 and 16) were used. The numbers above
lanes 1-4 and 9-12 correspond to the RNA
sequences depicted in Fig. 1. The sequences of the randomly
picked RNAs (A, B, C, and Mix) are
not depicted. B, purified GST-TIA-1 or GST-TIAR was incubated
with
P-labeled selected RNA 1-1 (lanes
1-9) or R-2 (lanes 10-18) in the presence of
increasing amounts of unlabeled competitor RNA (selected RNA 1-1,
selected RNA R-2, or the mixture of 20 RNAs picked randomly from the
library) as indicated. UV-cross-linking and subsequent analysis was
performed as in A. The unlabeled competitor RNA was present in
10-, 20-, 40-, or 60-fold molar excess compared with labeled RNA. C, purified GST-TIA-1 was incubated with
P-labeled selected RNA 1-1 in the presence of increasing
amounts of unlabeled competitor RNA (selected RNA 1-1, truncated
selected RNA 1-1, or mutated/truncated selected RNA 1-1) as indicated.
The unlabeled competitor RNA was present in 10-, 20-, or 60-fold molar
excess compared with labeled RNA.
Figure 3: Schematic diagram of TIA-1 and TIAR mutants. Fusion proteins between GST and different combinations of the RNA binding domains (RRMs) were constructed for TIA-1 and TIAR as described under ``Experimental Procedures.'' The structural domains of the wild-type proteins include the three RRMs, the RNP 1 and RNP 2 motifs, and the glutamine-rich carboxyl region. The sequence of each RNP 1 and RNP 2 motif is depicted. In each case, the upper sequence is that of TIA-1, the lower sequence is that of TIAR.
Figure 4:
UV-cross-linking of TIA-1 and TIAR
truncation mutants to RNA. Purified GST-TIA-1 (upper panel) or
GST-TIAR (lower panel), and each of the RRM mutants depicted
in Fig. 3was UV-cross-linked to [P]RNA
as described under ``Experimental Procedures.'' The
[
P]RNA was either selected RNA (S) or a
mixture of 20 RNAs picked randomly from the library (R). The
selected RNA used in the cross-linking studies with the TIA-1 mutants
was sequence 1-1; the selected RNA used with the TIAR mutants was
sequence R-2 (see Fig. 1). The migration of molecular mass
markers is shown at the right.
To assess
the strength of the interaction between full-length TIAR or TIAR RRM
truncation mutants and the selected RNA sequence, nitrocellulose filter
binding assays were performed. Fig. 5shows the normalized
binding curves for the full-length GST-TIAR protein and several of the
RRM truncation mutants. Although neither RRM 1 nor RRM 3, when
expressed in isolation, bind to the selected RNA sequence in this assay
even when present in micromolar concentrations (data not shown), the
affinity of RRM 2 for the selected RNA sequence is higher when RRM 2 is
expressed together with RRM 1 and RRM 3 (K = 2
10
M) rather than
alone (K
= 5
10
M). Consistent with the UV-cross-linking studies, the
TIAR constructs did not bind to the random mixture of in vitro transcribed RNAs (data not shown). The filter binding data,
together with the UV-cross-linking results, suggest that the second
RNA-binding domains of TIA-1 and TIAR contain the determinants of
sequence-specific binding. However, at least for TIAR, additional
structural domains of the protein appear to contribute to the affinity
of the protein-RNA interaction.
Figure 5:
Kinetics of binding of TIAR RRM
2-containing mutants to the selected RNA. Nitrocellulose filter binding
assays were performed to measure the affinity of TIAR and mutants
containing RRM 2 either alone or in combination with RRMs 1 and/or 3
for selected RNA R-3 (see Fig. 1). Each point represents the
average of three independent binding reactions. The data for each curve
are normalized to the saturation point for the RNA and corrected for
the fraction of protein active in RNA binding as described under
``Experimental Procedures.'' The legend to the data points is
shown in the upper left corner of the plot. The Kvalues shown below the plot are equal
to the protein concentrations at which 50% of RNA is
bound.
Figure 6:
Interaction between TIA-1/R RRMs and
cellular RNA. Total RNA extracted from PO
-labeled Jurkat cells was incubated with
equal molar quantities of GST-TIA-1, GST-TIAR or truncation mutants of
each of these proteins, and affinity- precipitated with
glutathione-Sepharose. The precipitated RNA was purified, separated on
1.2% formaldehyde agarose gels, and transferred to nitrocellulose. The
identity of each protein is indicated at the top of each lane. The
lanes labeled ``Total RNA'' show a fraction (approximately
1/20) of the RNA that was used in each binding experiment. The
migration of DNA size markers is shown at the
right.
We have employed in vitro selection/amplification
from pools of random RNA sequences to identify those RNA sequences for
which the TIA-1 and TIAR proteins have the highest affinity. After five
rounds of selection, 90% of the RNA sequences selected by both proteins
contain short stretches of uridylate residues ranging in length from 3
to 11 nucleotides. The sequences do not appear to have similarity with
regard to the distance between the uridylate stretches, the regions
flanking the uridylate stretches, or the predicted secondary structure.
UV-cross-linking experiments confirm that TIA-1 and TIAR bind the
selected sequences, that the interaction is specific, and that the
uridylate stretch is required for the interaction (see Fig. 2).
The affinity of the interaction (K of 8 nM as determined by filter binding assays) is similar to that
observed for other RNA-binding proteins (such as the hnRNP A1 protein)
and their selected RNA sequences(15) . Given the 80-90%
amino acid identity between the RNA binding domains of TIA-1 and TIAR,
it is not surprising that that the two proteins selected very similar
RNA sequences.
Although TIA-1 and TIAR each contain three RRM-type RNA-binding domains, our results suggest that the second domain of each of these proteins mediates the sequence-specific binding. Mutational analysis demonstrates that RRM 2 is both necessary and sufficient for binding to the RNAs selected by the full-length protein. Studies of other RRM-type RNA-binding proteins (hnRNP C, U1A, and U1 70K), have suggested that their RNA binding specificities are dependent on amino acids immediately carboxyl-terminal to the RRM domain(13, 16, 17) . Our results are not inconsistent with this, since the 15 amino acids linking the second and third RRMs are present in each mutant that contains RRM 2. A single RRM of a multi-RRM protein having the same sequence specificity as the full-length protein is not unique to the TIA-1 and TIAR proteins. The U1 snRNP A protein has two RRMs, only one of which appears to be required for binding to U1 RNA(18, 19) . Similarly, the third RNA binding domain of Hel-N1, like the full-length protein, binds to the 3` untranslated region of c-myc mRNA(20) . An analysis of the binding specificity of the hnRNP A1 protein, on the other hand, shows that its two RRMs have sequence specificities which differ both from each other and from the full-length protein(15) . Thus, within the RRM family of RNA-binding proteins, there does not appear to be uniformity with regard to the contribution of individual RRMs to overall binding specificity.
Although the third RNA-binding domains of TIA-1 and TIAR do not bind to the uridylate-rich RNAs selected by the full-length protein, they clearly do have RNA binding activity as illustrated by their ability to affinity-precipitate cellular RNA (see Fig. 6). The ability of RRM 3 to bind to cellular RNA but not to the mixture of random RNAs in vitro transcribed from the oligonucleotide library (see Fig. 4) may be due to differences in the sensitivities of the assays. It is quite possible that RRM 3 interacts with specific RNA sequences (that differ from the uridylate stretches) but that these sequences were not identified using the selection/amplification method because they are of low affinity or because, as has been demonstrated for U2 snRNP-B" binding to U2 RNA, the interactions require additional proteins (21, 22, 23) .
In contrast, we have been
unable to demonstrate any RNA binding activity for the first
RNA-binding domain of either TIA-1 or TIAR. The RNP 1 octamer of the
first RNA-binding domains of TIA-1, TIAR, and homologous proteins found
in Drosophila(8, 9) and C. elegans(10) are all somewhat atypical in that they contain
aspartic acid in the first position (see Fig. 3for human TIA-1
and TIAR RNP 1 sequences). The RNP 1 octamers of the second and third
RNA-binding domains of the TIA proteins each have a lysine in the first
position and thereby resemble the RNP 1 sequences of the majority of
RRM-type proteins which contain positively charged amino acids (either
arginine or lysine) in that position(5, 24) . It has
been proposed that the four-stranded antiparallel -sheet within
the RNA-binding domain of RRM-type proteins provides a general surface
for RNA binding, and that the highly conserved basic and aromatic amino
acids of RNP 1 and 2 (which form two of the
-strands) are involved
in direct interactions with
RNA(5, 13, 24, 25) . The first
residue of the RNP 1 octamer of the U1A RRM 1 has been shown to be
critical for U1A binding to U1 RNA; replacement of the arginine with
the uncharged glutamine completely abolished binding to U1
RNA(26, 27) . One other RRM-type protein with a
negatively charged amino acid in the first position of RNP 1 is
ASF/SF2. The ASF/SF2 RRM 2 RNP 1 octamer begins with an aspartic acid
(although there appears to be debate as to whether the eight amino acid
sequence beginning with aspartic acid, or an eight amino acid sequence
three residues downstream and beginning with a glycine, is the actual
RNP 1 sequence)(24, 28) . Additional studies are
necessary to determine whether the negatively charged aspartic acid of
TIA-1/R RNP 1 affects the RNA binding activity of RRM 1. It is possible
that RRM 1 may preferentially interact with RNA that has been modified
in ways which decrease its net negativity (e.g. by capping).
Uridylate stretches, frequently found in regulatory regions of RNAs
such as the 3` splice site of introns (29, 30) and 5`
and 3` untranslated regions(20, 31) , appear to be
common targets of RRM-type RNA-binding proteins. The hnRNP C and Hel-N1
proteins both selected sequences containing short uridine stretches
when similar selection/amplification approaches were used with these
proteins(13, 20) , and the Sex-lethal (Sxl) protein of Drosophila has been shown to interact with poly(U) sequences
in transformer (tra) and Sxl
pre-mRNAs(32, 33, 34, 35, 36) .
Because cells contain a multitude of RNA-binding proteins, some of
which may compete with TIA-1 and TIAR for binding to RNA, it is
important to characterize the RNA binding activity of TIA-1 and TIAR
not only in vitro but also in cells. The similarity between
the uridylate stretch consensus sequences identified for the TIA-1/R
and hnRNP C proteins, together with the size distribution of the
cellular RNAs interacting in vitro with TIA-1/R, suggested to
us that the TIA proteins may associate in cells with pre-mRNA or mRNA.
Although we have not been able to demonstrate an in vivo interaction between endogenous TIA-1 or TIAR and RNA, both of
these proteins can be isolated by oligo(dT) affinity chromatography of
lysates prepared from UV-irradiated TIA-1 or TIAR COS cell
transfectants ()indicating that, at least when
overexpressed, the proteins interact in cells with polyadenylated
RNA(37, 38) .
The identification of uridylate stretches as high affinity binding sites for TIA-1 and TIAR, together with the demonstration of in vivo interactions between these proteins and polyadenylated RNA, suggest that the TIA proteins might interact with regulatory regions of RNA transcripts to modulate gene expression. It is not known how the RNA binding activities of these proteins relate to their effector functions in apoptotic cell death. An intriguing possibility is that the TIA proteins regulate the processing or translation of RNA transcripts encoding mediators of apoptosis. Alternative splicing of ICE(39) , Ich-1(40) , and bcl-x (41) can produce mRNAs encoding both positive and negative regulators of apoptosis. Studies aimed at determining whether the TIA proteins bind to uridine-rich regions of such RNAs are under way.