Analysis of the 25 Arabid-opsis Pumilio APUM proteins presenting PUF repeats reveals that 12 APUM-1 to APUM-12 have a PUF domain with 50–75% similarity to the Drosophila PUF domain.. Thr
Trang 1proteins – binding specificity and target candidates
Carlos W Francischini and Ronaldo B Quaggio
Departamento de Bioquı´mica, Instituto de Quı´mica, Universidade de Sa˜o Paulo, Brazil
Introduction
The translational control of RNA is an important
reg-ulatory process in animal development This regulation
is accomplished by sequence-specific RNA-binding
proteins that recognize cis-acting elements usually
located in the 3¢ UTR In recent years, and as a result
of great efforts aiming to understand the mechanism
of RNA control in animals, the function of a diverse
number of RNA-binding proteins has been elucidated
[1–4] Despite this, translational control through the
binding of RNA-binding proteins to 3¢ UTR
tran-scripts has been poorly described in plants
PUF proteins are a large family of RNA-binding
proteins found in all eukaryotes These proteins reduce
the expression of mRNA targets by binding in 3¢ UTR regulatory elements, thus controlling translation or mRNA stability [5] Members of the PUF family have been implicated in diverse processes in development In Drosophila, Pumilio binds to the Nanos response ele-ment (NRE) sequence within the 3¢ UTR of maternal hunchback mRNA and reduces its expression in the posterior pole of the embryo This control is essential for abdomen formation [6] In Caenorhabditis elegans hermaphrodites, the Pumilio homolog FBF binds to the 3¢ UTR of fem-3 mRNA, repressing its translation and controlling the sperm–oocyte switch [7] Dictyoste-lium PufA represses pkaC mRNA and inhibits the
Keywords
Arabidopsis; PUF proteins; RNA-binding
protein; three-hybrid system; translational
control
Correspondence
R B Quaggio, Instituto de Quı´mica,
Departamento de Bioquı´mica, Universidade
de Sa˜o Paulo, Avenida Professor Lineu
Prestes, 748, Sa˜o Paulo 05508-000, Brazil
Fax/Tel: +55 11 3091 2171
E-mail: rquaggio@iq.usp.br
(Received 24 March 2009, revised 15 July
2009, accepted 22 July 2009)
doi:10.1111/j.1742-4658.2009.07230.x
PUF proteins regulate both stability and translation through sequence-spe-cific binding to the 3¢ UTR of target mRNA transcripts Binding is medi-ated by a conserved PUF domain, which contains eight repeats of approximately 36 amino acids each Found in all eukaryotes, they have been related to several developmental processes Analysis of the 25 Arabid-opsis Pumilio (APUM) proteins presenting PUF repeats reveals that 12 (APUM-1 to APUM-12) have a PUF domain with 50–75% similarity to the Drosophila PUF domain Through three-hybrid assays, we show that APUM-1 to APUM-6 can bind specifically to the Nanos response element sequence recognized by Drosophila Pumilio Using an Arabidopsis RNA library in a three-hybrid screening, we were able to identify an APUM-binding consensus sequence Computational analysis allowed us to identify the APUM-binding element within the 3¢ UTR in many Arabidopsis tran-scripts, even in important mRNAs related to shoot stem cell maintenance
We demonstrate that APUM-1 to APUM-6 are able to bind specifically to APUM-binding elements in the 3¢ UTR of WUSCHEL, CLAVATA-1, PINHEAD⁄ ZWILLE and FASCIATA-2 transcripts The results obtained
in the present study indicate that the APUM proteins may act as regulators
in Arabidopsis through an evolutionarily conserved mechanism, which may open up a new approach for investigating mRNA regulation in plants
Abbreviations
APBE, APUM-binding element; APUM, Arabidopsis Pumilio; IRP, iron regulatory protein; NRE, Nanos response element.
Trang 2development of fruiting bodies [8], whereas, in yeast,
both Puf3 and Puf5 (Mpt5) proteins promote the
decay of COX17 and HO mRNA, respectively,
through binding to their 3¢ UTR sequences [9,10]
Although members of this family of proteins have
been shown to play distinct roles in different
organ-isms, the maintenance and self-renewal of stem cells
appears to be an ancestral function [5,11] Drosophila
Pumilio binds to a NRE-like sequence within the
3¢ UTR of cyclin B1, repressing its translation and
promoting germline stem cell development [12–14]
C elegansFBF also controls germline stem cell
main-tenance by regulating gld-1 mRNA expression and
sus-taining mitosis [15] The Planaria PUF homolog
DJPumis expressed in neoblasts, which are capable of
self-renewal and differentiation during Planaria
regen-eration DJPum inactivation by dsRNA was found to
cause a dramatic reduction in the number of neoblasts
and impaired tissue regeneration [16] In mammals,
PUM2 is expressed in human germline stem cells [17],
whereas the mouse homologs Pum1 and Pum2 are
expressed in fetal and adult hematopoietic stem cells,
as well as in fetal neural stem cells [11]
The canonical PUF domain comprises eight PUF
repeats of approximately 36 amino acids each,
arranged in tandem to form a single concave structure,
usually located in the C-terminal region of the protein
Each repeat is formed by three a-helices that align
with the equivalent helices in the adjacent repeat,
forming three ladders of helices running through the
domain [18,19] The crystal structure of the human
Pumilio⁄ NRE complex demonstrated that each repeat
of the PUF domain recognizes a single nucleotide in
the RNA Sequence-specific recognition is mediated by
three conserved amino acids residues present at
posi-tions 12, 13 and 16, located in the second helix of each
repeat [20] Recently, it was shown that these residues
are also important for C elegans FBF specificity,
suggesting that PUF proteins of different organisms
recognize RNA with the same modularity [21]
Although PUF proteins have been shown to
regu-late distinct mRNA targets across species, the
nucleo-tides recognized appear to be conserved because all
known mRNAs regulated by these proteins contain a
UGURN1-3AU(A⁄ U) sequence [7–10,15,22–29] In
addition to its ability to bind RNA, the PUF
domain was demonstrated to take part in the
pro-tein–protein contacts necessary for RNA regulation
[6,30,31]
In the present study, we report the first analysis of
plant proteins possessing PUF repeats Using
compu-tational analyses and yeast three-hybrid assays, we
found that at least six Arabidopsis thaliana proteins
possess eight PUF repeats and can specifically recog-nize the NRE sequence of Drosophila hunchback mRNA Through a yeast three-hybrid screening using
an Arabidopsis RNA hybrid library, we identified mRNAs that may be target candidates of Arabidopsis Pumilio (APUM) regulation The screen also allowed
us to determine a consensus sequence recognized by the six APUM proteins that can bind to the NRE sequence (APUM-1 to APUM-6) Using this consen-sus, we show that APUM proteins are able to bind to the 3¢ UTR of transcripts related to self-renewal and stem cell maintenance in the shoot apical meristem Moreover, the consensus sequence suggests that a great number of Arabidopsis transcripts are potential targets for regulation by the PUF family of proteins The results obtained reveal a molecular conservation of PUF proteins in Arabidopsis thaliana and suggest that translational regulation via binding to 3¢ UTR in plants may have a role as important as that previously described in animals
Results
PUF proteins in A thaliana blast-p analysis of the Arabidopsis genome database (The Arabidopsis Information Resource – TAIR; http://www.arabidopsis.org) with Drosophila Pumilio was carried out to localize PUF proteins Further pfam analyses using a cut-off E-value of 1.0 over the blast-p output identified 25 proteins containing PUF repeats, which is the largest number of putative PUF proteins found in a single organism to date We named the putative A thaliana Pumilio homologs APUM-1 to APUM-25 (Fig 1) clustal w alignment
of these protein sequences was used to generate a phylogenetic tree [32], indicating that they may be separated into four distinct groups of similar pro-teins, which we named groups I, II, III and IV (Fig 1A) Only 3 out of 25 proteins were found to fall outside these groups Within each group, some proteins show a degree of primary sequence identity, from 40% to 90% along their entire lengths, and from 63% to 96% among their PUF domains (Table 1) The analysis also showed that the PUF domain of the six proteins from group I are highly similar (approximately 50% identical and 75% simi-lar) to the Drosophila PUF domain (Fig 2A and Table 2), whereas the six proteins from group II have lower levels of similarity (30% identical and 50% similar) with the Drosophila PUF domain (Fig 2B and Table 2) Moreover, these 12 putative APUM proteins from groups I and II have eight PUF
Trang 3repeats in the C-terminal region (Fig 1B), equivalent
to the number found in the well-characterized PUF
proteins [5,11] Proteins from group III, group IV
and the three outsiders show more similarity among themselves than they do with the Drosophila PUF domain (data not shown)
A
B
Fig 1 Analysis of the 25 putative APUM proteins (A) Phylogenetic tree constructed based on CLUSTAL W alignment of all putative APUM proteins and Drosophila Pumilio (accession number A46221) Numbers rep-resent the bootstrap analysis from 1000 trials (B) Number of PUF repeats identified for each APUM in the PFAM analysis Gray circles represent the localization of repeats
in the protein and the numbers indicate the position of each repeat in the PUF domain Black circles represent repeats identified in the PFAM that fall outside the C-terminal region APUM proteins were named APUM-1 to APUM-25.
Trang 4Prediction of APUM-binding specificity
In the human Pumilio–NRE complex, residues 12 and
16 of each repeat make hydrogen bonds or Van der
Waals contacts with a specific RNA base, whereas
res-idue 13 makes stacking interactions [20] An analysis
of these residues for each PUF repeat of all putative
APUM proteins showed that APUM-1 to APUM-6
have the amino acids in positions 12, 13 and 16,
simi-lar to human and Drosophila Pumilio On the other
hand, the amino acids in these positions in APUM-7
to APUM-12 are more similar to those found in yeast Puf4 and Puf5 proteins (Table 3; data not shown) The remaining APUM proteins do not show conserva-tion of residues 12, 13 and 16 with any well-character-ized PUF homolog (data not shown) and possess less than eight PUF repeats in their PUF domains (Fig 1B)
The analysis allows us to suggest that the group I proteins (APUM-1 to APUM-6) share the same RNA-binding specificity of Drosophila and human Pumilio-1 Thus, we expected that these APUM proteins should bind to the NRE sequence within the 3¢ UTR of Drosophila Pumilio mRNA target hunchback The group II proteins APUM-7 to APUM-11, which have a nonconservative Asnfi His substitution in residue 13 of repeat 7 (Table 3), would be expected to have binding to the second nucleotide in the UGU triplet impaired Binding to this nucleotide is essential for RNA recognition [24,25,33–36]
Table 1 Amino acid identity between some putative Arabidopsis
PUF proteins in the full-length and PUF domain.
Gene ID Similar to:
Full protein identity (%)
PUF domain identity (%) At2g29190 At2g29140 ⁄ At2g29200 90 95
A
B
Fig 2 CLUSTAL W alignment of the APUM proteins with the PUF domains most similar to Drosophila PUF domain (A) APUM proteins of group I (B) APUM proteins of group II.
Trang 5Binding of APUM to NRE
To test the predictions regarding the RNA-binding
specificities of the putative APUM proteins, we
investi-gated the capacity of APUM to bind to the NRE
sequence of hunchback mRNA (Fig 3B) [36] We used
the APUM-2 protein as a representative member of
group I proteins and APUM-7 as a representative of
group II APUM proteins (Table 3)
Protein–RNA interactions was evaluated using yeast three-hybrid system, which was shown to be a reliable approach for identifying true interactions [25,33,37–39] This system uses LexA⁄ MS2 coat protein fusion to tether the RNA hybrid to the promoter of reporter genes The RNA-binding protein is produced as a tran-scription activation fusion domain through which the reporter genes (HIS3 or LacZ) are transcribed when the RNA–protein interaction is established (Fig 3A) [37] The pYESTrp3⁄ APUM-2 and pYESTrp3 ⁄ APUM-7 vectors were transformed in the yeast YBZ-1 strain [40] together with the pRH5¢ ⁄ NRE vector After growth in selective medium, individual colonies were tested for LacZ reporter activation The results showed that LacZ reporter was activated in yeast colonies transformed with APUM-2 and NRE, but not with APUM-7 and NRE (Fig 3C)
In the NRE fragment, two UGU sequences, named Box A and Box B (Fig 3B), were shown to be essential for Drosophila Pumilio recognition because UGU resi-dues substitution in both boxes abolished the interaction with the protein [36] To verify the specificity of
APUM-2 for the NRE, three mutant NREs with nucleotide sub-stitutions in the UGU sequence of Box A, NRE(A)B+); Box B, NRE(A+B)); and in both Box A and B, NRE(A)B)), were used as baits in the yeast three-hybrid assay (Fig 3B) [33] The results of reporter activation indicated that APUM-2 interacts with NRE(A)B+),
Table 2 The 12 Arabidopsis PUF domains most similar to the
Dro-sophila PUF domain.
Gene ID
Similarity to Drosophila PUF domain (%)
Identity to Drosophila PUF domain (%)
Table 3 Alignment of the nucleotide binding residues of human Pumilio-1 and the corresponding residues in the APUM proteins The well-characterized Drosophila Pumilio and yeast Puf4 are also shown Amino acids at position 12, 13 and 16, respectively, of each repeat are boxed in gray.
a
Well-characterized Drosophila Pumilio and yeast Puf4 proteins included as comparison.bPreferential sequences recognized by Drosophila Pumilio [27] c Preferential sequences recognized by yeast Puf4 [23].
Trang 6whereas no interaction was observed with NRE(A+B)) and NRE(A)B)) (Fig 3C) Quantitative analysis of LacZ expression showed that the binding affinity of APUM-2 for NRE(A)B+) was not significantly altered with respect to the wild-type NRE sequence, whereas the interaction with NRE(A+B)) and NRE(A)B)) was fully abolished (Fig 3D) Furthermore, assays using APUM-7 as prey did not interact with the wild-type or any of the mutant NREs (Fig 3C)
To confirm that the result of binding specificity observed between APUM-2 and NRE can be extended
to the remaining group I proteins, we tested the interac-tion of APUM-1, APUM-3, APUM-4, APUM-5 and APUM-6 with wild-type and mutant NREs Qualitative (data not shown) and quantitative analysis of LacZ activity (Fig 3D) revealed that all five APUMs tested recognized the NRE and NRE(A)B+) sequences, but did not bind to NRE(A+B)) or NRE(A)B))
Together, these results confirmed our predictions regarding the binding specificity of the subset of group
I APUM proteins, showing that A thaliana has at least six PUF proteins with conserved RNA-binding and similar specificity The group I APUM proteins recog-nize Box B within the NRE sequence because UGU substitutions in Box A did not abolished the interac-tion The Box B sequence presents a trinucleotide AUA downstream of the UGU motif (Fig 3B), indicating that APUM proteins could recognize the sequence UGUANAUA, as do PUF proteins of other organisms (Table 3) [11] These observations allow us to speculate that, although NRE is not the natural RNA target in Arabidopsis, the Box B sequence should mimic the authentic Arabidopsis targets On the other hand, APUM-7 was unable to bind to NRE, possibly as a result of the nonconservative substitution at repeat 7
Influence of the Aspfi His substitution on the APUM-7 RNA-binding capacity
The APUM-7 protein has the same binding residues as yeast Puf4 and Puf5 (Table 3; data not shown), except for an Aspfi His substitution at repeat 7 The yeast proteins have been shown to recognize sequences similar
to the NRE Box B sequence (Fig 3B and Table 3) [23]
We considered that, if APUM-7 did not bind to NRE because of the nonconservative substitution at residue 13 of the repeat 7, then changing this back to Asp may restore APUM-7 binding to NRE In a simi-lar manner, if this Asp is critical for interaction, its substitution for a His would be expected to abolish binding of APUM-2 to NRE
To evaluate these hypotheses, we tested the interaction
of APUM-2⁄ N fi H (APUM-2 with the Asp fi His
A
B
C
D
E
Fig 3 Interaction analysis between APUM and the NRE transcript.
(A) Schematic representation of the yeast three-hybrid system (B)
Sequence of the wild-type NRE transcript (WT) and NRE mutants
with nucleotides substitutions in Box A, NRE(A)B + ); Box B,
NRE(A+B)); and in both Box A and B, NRE(A–B–) (C) Qualitative
analysis of LacZ reporter activation in the interaction of APUM-2
and APUM-7 with NRE WT and NRE mutants The iron responsive
element RNA and the IRP protein were used as positive controls
for the interaction (D) Quantitative analysis of LacZ reporter
activa-tion in the interacactiva-tion between APUM-1, APUM-2, APUM-3,
APUM-4, APUM-5 and APUM-6 with NRE WT and mutants (E)
Interaction assay of APUM-2 with Asn to His substitution in the
residue 13 of the repeat 7 (APUM-2 ⁄ N fi H) and the protein
APUM-7 with His fi Asn substitution in the same position
(APUM-7 ⁄ H fi N) with the NRE transcript.
Trang 7substitution at residue 13 of repeat 7) and of
APUM-7⁄ H fi N (APUM-7 with the His fi Asp substitution at
residue 13 of repeat 7) with the NRE transcript in the
three-hybrid system The results obtained showed that
APUM-2⁄ N fi H continued to recognize the NRE,
whereas APUM-7⁄ H fi N did not (Fig 3E), indicating
that the failure to bind to NRE is not a result of
Aspfi His substitution
Because APUM-8 to APUM-11 proteins share the
same substitution in repeat 7, it is expected that they
will behave as APUM-7 does (i.e they will not bind to
NRE) Similarly, the APUM-12 protein has exactly the
same amino acids binding residues as
APUM-7⁄ H fi N, which suggests that they may exhibit
simi-lar binding behaviors
Yeast three-hybrid screen to identify
APUM-binding RNA
To identify putative mRNA targets of APUM
pro-teins, we used a yeast three-hybrid screen, which was
shown to be a useful and reliable approach for
profil-ing mRNAs that bind directly to a specific
RNA-bind-ing protein [41–43] AccordRNA-bind-ingly, we generated an
Arabidopsis RNA hybrid library of small fragments
(50–150 bp) and used this as prey in a three-hybrid
screen with APUM-2 as bait (Fig 4A)
From approximately eight million independent
Ara-bidopsisRNA sequences screened, 189 positive
interac-tions derived from 63 distinct sequences were isolated
(Fig 4B) Of these 63 clones, 27 (43%) were insert
cloned in antisense position The other 36 clones
(57%) were sense sequences, with five (14%) 3¢ UTR
transcripts (Fig 4C and Table 4)
Computational analysis of RNA sequences
identified in the yeast three-hybrid screen
Although only five of 63 transcripts identified by the
three-hybrid screens were derived from 3¢ UTR
regions, all of them (sense and antisense) bound to bait
specifically, suggesting the existence of a consensus
motif within these 63 distinct transcripts recognized by
APUM-2 We therefore analyzed these sequences using
multiple expectation maximization for motif elicitation
(meme) as a motif discovery tool [44] (http://meme
nbcr.net/meme/intro.html) The analysis identified an
eight nucleotide motif present in all 63 transcripts
(Fig 5A) The consensus possesses a UGUR
tetranu-cleotide sequence, which has been reported to be
pres-ent in all targets of the PUF family [5,11] In addition,
a (A⁄ U)(U ⁄ G)(A ⁄ U ⁄ C) sequence located one
nucleo-tide downstream of the UGUR motif is highly similar
to the trinucleotide AUA and AUU present in the target consensus of many other PUF members [21,23,27,39,45] In some transcripts, these last three nucleotides were AGA and AGC, which have not been described for any other PUF protein to date
On the basis of these results, we were able to iden-tify two NRE Box B-like consensus sequences, which
we named the APUM-binding elements (APBE) (Fig 5B) The APBE of the 3¢ UTR sequences identi-fied in the screening is shown in Table 4
Evaluation of the APBE identified by means of yeast three-hybrid screen
Because the deduced binding consensus is very small,
it must be present in a large number of Arabidopsis transcripts Indeed, a search for the APBE motif in all 5¢ UTR, 3¢ UTR and ORFs annotated at the TAIR database showed that approximately 56% of all ORF
A
B
C
Fig 4 Screen of an Arabidopsis RNA hybrid library to identify RNA bound by APUM-2 (A) Scheme of the three-hybrid strategy used in the screen (B) Number of colonies identified in each step of the screen (C) Distribution of the 63 distinct sequences in relation of their position in the Arabidopsis transcriptome.
Trang 8sequences and 43% of all 3¢ UTR sequences have at
least one binding consensus for APUM proteins,
whereas, in 5¢ UTR, its occurrence is significantly
lower (Table 5)
As a result of the high occurrence of the APUM
binding sites in the plant genome, we decided to focus
in the binding of APUM consensus to 3¢ UTR
tran-scripts expressed in the tissue related to plant
meris-tems because the regulation of transcripts related to
stem cell maintenance is considered to be an ancestral
function of PUF proteins in animals Thus, a 32
nucle-otide region of the 3¢ UTR of CLAVATA-1(CLV-1)
(At1g75820), ZWILLE⁄ PINHEAD (ZLL) (At5g43810),
WUSCHEL (WUS) (At2g17950) and FASCIATA-2
(FAS-2) (At5g64630) transcripts was cloned in the
pRH5¢ vector and tested with APUM-2 protein in the
three-hybrid system (Fig 5C) These four transcripts
have been described to code for proteins involved in
diverse developmental processes, including shoot
meri-stem organization, meri-stem cell maintenance and
mainte-nance of cellular organization of apical meristems [46–
50] The LacZ reporter was activated in all assays
tested (Fig 5D), indicating that the APBE motif is
sufficient for APUM-2 recognition The APUM-1,
APUM-3, APUM-4, APUM-5 and APUM-6 proteins
also interacted with these transcripts, whereas
APUM-7 did not (data not shown) These results confirm that
the APBEs can be recognized by proteins of group I
and also indicate that these consensus can be useful to
identify putative mRNAs targeted by APUM-1 to
APUM-6
Group I APUM proteins requires nucleotides in
both 5¢ and 3¢ of the APBE motif
In the computational analysis used to identify a
con-sensus binding motif, no biases towards nucleotides
outside the APBE were identified (Fig 5A,B)
How-ever, the interactions between APUM-2 with the NRE
transcript and with the four 3¢ APBE UTR sequences chosen by bioinformatics analysis showed distinct val-ues of LacZ reporter activation (Figs 3D and 5E) These data suggest that binding affinity may be influ-enced either by nucleotides outside of the consensus motif or by small variations within the consensus The interaction of APUM-2 with the FAS-2 tran-script was the strongest among the interactions tested
in the three-hybrid system (Figs 3D and 5E) The FAS-2transcript used in the binding assay differs from that of WUS, CLV-1 and ZLL sequences in both APBE and flanking nucleotides (Fig 5C), whereas its binding core element is exactly the same as that of Box B present in the NRE transcript (Fig 3B) Because APUM-2 binds to FAS-2 approximately five-fold more strongly than to NRE (Fig 5E), we can sug-gest that specific nucleotides flanking the core element
of FAS-2, which are not present in the NRE sequence, may contribute to APUM-2 binding
To examine the contributions of flanking nucleotides
in the affinity between APUM-2 and FAS-2, we pro-duced double mutations in nucleotides upstream and downstream of the APBE (Fig 6A) Quantification analysis of b-galactosidase activity showed that several substitutions reduced the binding affinity to different degrees (Fig 6B,C) Most significantly, mutations at positions )1 ⁄ )2 abolished the interaction with APUM-2 (Fig 6B) The interaction of APUM-1, APUM-3, APUM-4, APUM-5 and APUM-6 proteins with the FAS-2 transcript was also abolished when the nucleotides at positions )1 ⁄ )2 were substituted (Fig 6D)
These results demonstrate that nucleotides upstream and downstream of the binding consensus are critical for interaction with APUMs from group I We can therefore consider the APBE as the core binding element, whereas other flanking nucleotides contribute
to the accomplishment of strong or weak inter-actions
Table 4 3¢ UTR transcripts identified in the yeast three-hybrid screening Upper case letters and boxed sequences indicate the presumptive APUM binding sites Information about each gene product was obtained from the TAIR database.
Gene ID (number of
At3g63500 (7) Protein containing PHD domain;
unknown function
ugcgucugacaUGUACAGCcccugccaaauuuuaauaggcaat AGUAAAUAaauaacgacaagaagcaaaugg
At5g24490 (1) Ribosomal protein; unknown function cucaucucuccuuacaguuuaccuguguaggaguuaggguucuuga
auaaacaaugcaacaaagauuguagaagucagUGUACAUA At4g36040 (1) Protein containing DNAJ domain;
unknown function
cuacgucggacggaacugggaaaccgaucaguguugguagugaguuaa cucggugaccgaguuaguagaacgaguuaauuagUGUAAAUAcgaagcca At4g39090 (1) ‘Embryo defective’ (RD19); response to
physiological stress
uuuaucucugcuucuugcuUGUAAAUAaa At3g47470 (1) Chlorophyll a ⁄ b-binding protein cuccaugaacaaauuuggaaucuucaaUGUACAGA
Trang 9Multiple PUF members in A thaliana Currently, the largest number of PUF proteins found
in a single organism was in C elegans, which has eleven homologs, whereas yeast has six; human and mouse possess two; and Drosophila and Dyctiostelium have only one member [5] Recently, new studies have revealed the presence of ten, two and one homologs
in Trypanosome, Plasmodium and Planaria, respec-tively [16,33,51] In the present study, we showed that the A thaliana genome may contain the largest number of putative PUF proteins described to date (Fig 1)
Functional characterizations of different homologs have shown that a single PUF protein may be associ-ated with several distinct developmental processes Moreover, PUF proteins in the same organism may have overlapping and independent functions In C ele-gans, FBF-1 and FBF-2, which share 90% sequence identity, act redundantly in sperm–oocyte switch and germ stem cell maintenance [7,15] However, these two proteins show distinct patterning functions in the distal germ line, independently affecting the number of cells
in the mitotic region [29] Also in C elegans, the lack
of PUF-8, which is more similar to Drosophila Pumilio than to FBF, causes germ line dedifferentiation and the formation of fast growing tumors [52] In Drosoph-ila, the single Pumilio has been related to many inde-pendent processes [14,53–56], and five of the six yeast PUF homologs, which are significantly divergent in sequence, appear to have predominately distinct func-tions [23]
In A thaliana, we have identified three highly con-served gene families that account for 22 of 25 putative PUF proteins The three remaining proteins can be divided into a closely-related pair and a single outsider (Fig 1A) The large number of copies of highly similar proteins (Table 1) could be an indicative of redundant functions in the plant However, these functions might
be specific to each group of duplicated genes We
A
B
C
D
E
Fig 5 Identification and evaluation of a common sequence motif
in the mRNA obtained from yeast three-hybrid screen (A) Eight
nucleotide motif found by MEME analysis in all 63 distinct clones.
(B) Deduced APBE (C) Computational identification of an APBE
(boxed sequences) in the 3¢ UTR region of transcripts FASCIATA-2
(FAS-2), WUSCHEL (WUS), CLAVATA-1 (CLV-1) and ZWILLE ⁄
PIN-HEAD (ZLL) The sequences shown are the 3¢ UTR regions used in
the yeast three-hybrid assays (D) Qualitative analysis of LacZ
repor-ter activation in the inrepor-teraction between APUM-2 and the
tran-scripts FAS-2, WUS, CLV-1 and ZLL The NRE sequence (Fig 2B)
was used as a positive control (E) Quantitative analysis of LacZ
activity in the interactions shown in (D).
Table 5 Occurrence of the APBE in the A thaliana transcriptome.
ORFa
a Known and putative sequences in the A thaliana database (TAIR).
A total of 21835 3¢ UTR sequences, 20 564 5¢ UTR sequences and
36 690 ORFs were analyzed separately and independently of length.
Trang 10therefore predict that various PUF of A thalina may
be involved in many different processes in the plant
RNA-binding capacity of the APUM proteins
pfam analysis of all putative APUM proteins showed
that the six APUM group I proteins, all highly similar
to Drosophila Pumilio (Figs 1A and 2A and Table 2),
have the eight conserved repeats characteristic of the
PUF family of proteins (Fig 1B) These six homologs
have the same residues necessary to confer RNA
speci-ficity in human Pumilio-1 (Table 3) and can bind to
the NRE sequence specifically (Fig 3) Six group II
APUM proteins (APUM-7 to APUM-12) (Fig 1B)
also possess eight PUF repeats, some of which do not show conservation in residues directly involved in nucleotide recognition (Table 3) Through site-directed mutagenesis and interactions assays, we showed that this substitution is not responsible for the APUM-7 binding impairment (Fig 3E)
Although PUF proteins have been shown to recog-nize RNA through a UGUR tetranucleotide followed
by an AU(A⁄ U) sequence, the number of nucleotides between these two sequences is variable among different homologs For example, C elegans FBF recognize RNA that have the UGUR and AUA sequence sepa-rated by two nucleotides, whereas C elegans PUF-8, Drosophila and human Pumilio and yeast Puf3
Fig 6 Analysis of binding affinity between APUM-2 and the FAS-2 3¢ UTR transcripts with nucleotide substitutions upstream and down-stream of the APBE (A) Double substitutions in flanking nucleotides of APBE (lower case) Bold letters in the wild-type sequence indicate the APBE The first nucleotide of the motif is numbered base one The individual adenine to guanine substitution at nucleotide four was per-formed to confirm the deduced APBE, which admits a guanine in this position (Fig 5) (B) Quantitative analysis of LacZ reporter activation in the interactions between APUM-2 and the FAS-2 transcripts with substitutions in nucleotides upstream of the APBE (C) Quantitative analy-sis of LacZ reporter activation in the interactions between APUM-2 and the FAS-2 transcripts with substitutions in the nucleotides down-stream of the APBE (D) Quantitative analysis of LacZ activation in the interactions of APUM-1, APUM-3, APUM-4, APUM-5 and APUM-6 with the FAS-2 transcript wild-type (WT) and FAS-2 transcript with substitutions at positions )2 ⁄ )1.