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Previous Article | Next Article 
Molecular and Cellular Biology, February 2008, p. 1298-1312, Vol. 28, No. 4
0270-7306/08/$08.00+0 doi:10.1128/MCB.00936-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Department of Molecular and Cellular Biology and Howard Hughes Medical Institute, University of Arizona, Tucson, Arizona 85721-0106
Received 25 May 2007/ Returned for modification 6 July 2007/ Accepted 25 October 2007
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ABSTRACT |
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INTRODUCTION |
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Decapping is a critical node in the control of the life of an mRNA. Moreover, the processes of mRNA decapping and translation are mechanistically intertwined and appear to compete with each other, at least in yeast (14). For example, decreasing translation initiation by a variety of means increases the rate of mRNA decapping (28, 33, 39). Conversely, inhibition of translation elongation leads to a significant decrease in the rate of decapping (3). Moreover, coimmunoprecipitation experiments suggested that prior to decapping, an mRNA exits translation and then assembles into a translationally repressed messenger ribonucleoprotein (mRNP) complex (45).
Additional evidence for a discrete population of nontranslating mRNPs has been that nontranslating mRNAs and the decapping machinery accumulate in discrete cytoplasmic foci called P bodies (also referred as GW182 or Dcp bodies) (17, 25, 30, 40). P bodies have now been observed in yeast, insect cells, nematodes, and mammalian cells and contain various proteins involved in mRNA decay, including the decapping enzyme (Dcp1/Dcp2); activators of decapping, Dhh1, Pat1, Lsm1-7, and Edc3; and the exonuclease Xrn1 (2, 20, 34). Moreover, P bodies have been suggested to be functionally involved in mRNA decapping (17, 40), nonsense-mediated decay (41, 46), mRNA storage (6, 9), general translation repression (15, 23), microRNA-mediated repression (26, 29, 36), and, possibly, viral packaging (5). In these cases, the analysis of P bodies, translation repression, and decapping suggests that the process of translation repression and mRNA decapping involves an initial step wherein the mRNA ceases translation and assembles an mRNP capable of decapping and accumulation in P bodies. This initial step would then be followed by a subsequent decapping reaction, although the relationship between translation repression, P-body mRNP assembly, and actual catalysis of decapping remains unclear.
An important protein in the process of mRNA decapping and translation repression is Pat1. Yeast strains lacking Pat1 show the strongest defects in decapping of any mutant besides defects in the decapping enzyme Dcp1/Dcp2 (7, 8, 44). In addition, efficient translation repression during glucose deprivation and P-body assembly requires Pat1 (15, 23, 42). Moreover, the overexpression of Pat1 leads to a global repression of translation and accumulation of mRNAs in P bodies (15). Pat1 is also a conserved protein and is found in P bodies in Saccharomyces cerevisiae, Drosophila melanogaster, and mammalian cells as well (15, 20, 38, 40). Despite this central role in mRNA decapping, P-body assembly, and translation repression, the properties of the Pat1 and its functional interactions are unknown.
In this work, we analyze Pat1 function in Saccharomyces cerevisiae by deletion and functional analyses. Our results indicate that Pat1 contains two important functional domains: one that promotes translation repression and P-body assembly and another one that promotes mRNA decapping in a manner enhanced by the assembly of the transcript into a P-body mRNP. In addition, we provide evidence that Pat1 binds RNA and has numerous domain-specific interactions with mRNA decapping factors. These results indicate that Pat1 is an RNA binding protein and a multidomain protein that functions at multiple stages in the process of translation repression and mRNA decapping.
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MATERIALS AND METHODS |
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strains, grown at 30°C to an optical density at 600 nm (OD600) of 0.6, lysed in a 5 M urea-2% sodium dodecyl sulfate (SDS) solution, and then loaded onto an SDS-10% polyacrylamide gel. Electrophoresed samples were transferred onto a Protran nitrocellulose membrane (Whatman, NJ) and then probed with anti-Flag antibody (Sigma). RNA analysis. For mRNA decapping analysis of the yeast reporter mRNA MFA2pG, cells were grown to an OD600 of 0.3 to 0.4 in SC medium supplemented with the appropriate amino acid and 2% galactose at 30°C. Cells were harvested and total RNA was extracted as described previously by Caponigro et al. (12). RNA was analyzed by running 20 µg of total RNA on 6% urea-polyacrylamide gels. Northern analyses were performed using a radiolabeled oligonucleotide, oRP140, which is directed against the MFA2pG reporter mRNA (13). Quantitation of blots was performed using a Molecular Dynamics (Sunnyvale, CA) PhosphorImager. Loading corrections were done using oRP100, an oligonucleotide directed against 7S RNA, a stable RNA polymerase III transcript (11).
After repeating the assay with newly transformed strains on three separate occasions, we performed statistical analysis on the resulting quantitation. A Mann-Whitney nonparametric test was performed on the results to determine if a statistically significant difference was observed between the deletion constructs. This ranking-based test allowed us to group those constructs that showed differences from the pat1 strain with a P value of less than 0.02.
Microscopy. High-optical-density cultures were grown to an OD600 of 1.0 in the appropriate medium. Cells were washed in SC medium supplemented with the appropriate amino acids without glucose, resuspended in the same medium, incubated in a flask in a shaking water bath for 10 min, and then collected by centrifugation. In the case of the cells at the mid-log phase of growth, they were grown to an OD600 of 0.3 to 0.5 in the appropriate medium with glucose. An aliquot of cells was then resuspended in the same medium for observation by use of the fluorescent microscope. Cells were observed on a Leica DM-RXA fluorescent microscope equipped with a mercury xenon light source. Images were captured by use of a Retiga EX (Q Imaging) digital camera and manipulated using Metamorph 6.0 software.
For analysis of P bodies following galactose induction, cells were grown in minimal medium supplemented with 2% sucrose to an OD600 of 0.3 to 0.4. At this point, the cells were washed in minimal medium, split in to two equal subcultures: one subculture was resuspended in minimal medium plus 2% sucrose, while the other subculture was resuspended in minimal medium plus 2% galactose. Following 120 min of induction, aliquots of cells were concentrated on a tabletop centrifuge, resuspended in their respective media, and observed on a Leica DM-RXA fluorescent microscope.
Yeast two-hybrid analysis.
The two-hybrid fusion plasmids were constructed by PCR amplification of the open reading frame (ORF) of the indicated genes or the Pat1 ORF deleted for certain domains and inserted into pOAD or pOBD-2 by homologous recombination in yeast strains PJ694a and PJ694
as described previously (10, 27). PJ694a strains containing pOAD alone or pOAD containing full-length Lsm1, Edc3, or Dcp1 were mated with PJ694 strains containing pOBD-2 alone or pOBD-2 with full-length Pat1 or Pat1 deletions. Interactions were measured by β-galactosidase plate assays as well as 3-amino-1,2,4-triazol dilution series assays.
Translational repression analysis. In vivo [35S]methionine labeling was performed by growing cells in minimal medium containing 2% sucrose, harvesting 12 ml of cells at an OD600 of 0.3, and then resuspending cells in 20 ml minimal medium containing 2% sucrose or 2% galactose plus 0.25% sucrose for 120 min to induce Pat1 overexpression. Cells were then labeled by adding 120 µl cold methionine (10 µg/ml) and 3 µl [35S]methionine (Amersham). At the determined time points, 1-ml aliquots were added to 1 ml 20% trichloroacetic acid. Samples were heated at 95°C for 20 min, collected on Whatman glass microfiber paper, washed with 10% trichloroacetic acid and 95% ethyl alcohol, and placed in scintillation fluid. This solution was then quantified on a scintillation counter.
In vitro translation and RNA binding assays. In vitro translation of 1 µg of Pat1-containing plasmid was performed with the TNT quick-coupled reticulocyte lysate system (Promega) according to the manufacturer's instructions. In vitro translation was performed in the presence of 20 µCi of in vitro translation-grade [35S]methionine (Amersham). The binding of in vitro-translated, [35S]methionine-labeled Pat1 and Pat1 deletion constructs to RNA homopolymers was performed according to the following protocol. Poly(U)-Sepharose and protein G-Sepharose (Promega) were prepared according to the manufacturer's recommendations and resuspended at a concentration corresponding to 100 µg (dry weight) per ml in binding buffer (10 mM Tris [pH 7.4], 2.5 mM MgCl2, 100 mM KCl, and 0.5% Triton X-100). Twenty-five microliters of poly(U)-Sepharose or protein G-Sepharose was mixed with 10 µl of full-length Pat1 in vitro-translated product, 10 µl of the product at residues 10 to 254, 100 µl of the product at residues 254 to 422, and 100 µl of the product at residues 422 to 763. This solution was mixed in a total of 300 µl in the presence of 200 U of RNasin and 0.5 µg/ml of yeast tRNA as a nonspecific competitor and incubated at 4°C for 45 min. In competition experiments, poly(U) or poly(C) (Amersham) was added to the binding reaction mixtures at the concentrations indicated in the figures. Following the binding reaction, the Sepharose beads were washed four times in binding buffer, resuspended in sample buffer, boiled for 2 min, and loaded onto an SDS-12% polyacrylamide gel. Following electrophoresis, the gels were exposed to PhosphorImager screens, and quantitation of blots was performed using a Molecular Dynamics (Sunnyvale, CA) PhosphorImager.
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RESULTS |
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strain and tested for its effect on Pat1 function in a variety of assays. In addition, to maintain the proper context of translation initiation and termination, 10 amino acids from the N terminus and 34 amino acids from the C terminus were included in each construct. We also observed a single amino acid change in the yRP840 genome sequence compared to the Saccharomyces Genome Database sequence (V688D); however, this appears to have no functional consequences, since our PAT1-containing plasmids are functionally no different from wild-type PAT1 (data not shown).
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strains are temperature sensitive and are unable to grow at 35°C. Analysis of the various Pat1 deletions revealed that any construct lacking residues 254 to 422 (254-422) was unable to grow at 35°C (Fig. 1C and 2). In contrast, Pat1 variants lacking residues 10 to 254 or 422 to 763 were still able to grow at 35°C, although strains expressing Pat1 lacking residues 422 to 763 were slightly growth impaired at high temperatures. These results define the domain between residues 254 and 422 as being the critical part of Pat1 for growth at high temperatures.
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variant showing a defect in function would be due to the deletion protein being unstable and being present only at reduced concentrations compared to wild-type Pat1. In order to examine this possibility, we tagged the different Pat1 deletion variants with a Flag epitope at their N termini, which did not affect Pat1 protein function, and examined their levels by Western blotting. Four relevant observations were observed in these experiments. First, the 254-763, 422-763, 697-763, 10-422, 10-131, 254-422, and 422-697 variants were all expressed at levels similar to those of the full-length protein (Fig. 1D). Therefore, any phenotype arising from these deletions cannot be due to reduced levels of Pat1 (see below). Second, we observed that the 10-254 variant was consistently expressed at lower levels than the full-length protein (Fig. 1D and data not shown). Surprisingly, despite this reduced level of expression, this deletion had no effect on cell growth and has only a small effect on decapping efficiency (see below). These observations suggest that reductions in the level of Pat1 can be tolerated without strong phenotypes. Third, we observed that the Pat1 variants with large deletions (10-763, 131-763, and 10-697) were expressed at reduced levels (Fig. 1D), although this does not impact the conclusions drawn from variants with smaller deletions (see below). Finally, as previously noted (47), we observed that Pat1 and its deletion variants ran anomalously for their predicted molecular weights on SDS gels.
The domain between residues 254 and 422 is required for Pat1 to stimulate decapping, and two other domains enhance the ability of Pat1 to activate decapping.
A second phenotype of pat1 strains is a defect in the rate of mRNA decapping (7, 8, 21, 44). Thus, we examined how different deletions in Pat1 affected mRNA decapping in yeast. To this end, we analyzed the unstable MFA2pG reporter RNA, which contains a poly(G) tract within the MFA2 3' untranslated region (18). The poly(G) tract is an effective block of 5'-to-3' exonucleolytic decay resulting in the formation of a decay intermediate referred to as the poly(G) fragment. With this reporter mRNA, pat1 strains show two differences from wild-type strains, indicating their defect in mRNA decapping. First, because deadenylation precedes decapping, pat1 strains accumulate a population of deadenylated mRNAs (Fig. 3A, lane 1, compared to the wild type on CEN, lane 11). Second, due to the decreased rate of decapping, the levels of the poly(G) fragment, whose abundance can be related to the decapping rate of the mRNA, are decreased (11). Thus, to examine how different domains of Pat1 affected mRNA decapping, we examined which constructs led to the loss of accumulation of deadenylated full-length mRNAs and restored the production of the poly(G) fragment.
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in decapping efficiency (Fig. 3A, compare lane 12 to the wild type on CEN, lane 11). Second, residues 10 to 254 also had a small effect on decapping (10-254) (Fig. 3A), although this effect could be due to the reduced levels of this particular deletion protein, or this region might be able to enhance decapping in some manner (Fig. 1D). Third, residues 422 to 763 affect the efficiency of mRNA decapping since any deletion of this region, including the precise deletion of residues 422 to 763 (lane 5), gives a partial defect in mRNA decapping without affecting protein levels. However, it should be noted that the domain at residues 422 to 763 appears to act through the region at residues 254 to 422 since strains containing the region at residues 422 and 763 without the region at residues 254 to 422 still show an absolute block in decapping (Fig. 3A, lane 12, and B). Taken together, these results indicate that two regions of Pat1 can directly affect decapping, with residues 254 to 422 being absolutely required for Pat1 to stimulate decapping and residues 422 to 763 affecting the efficiency with which residues 254 to 422 can promote decapping. The region at residues 422 to 763 is responsible for the role of Pat1 in translation repression and P-body assembly. Pat1 has been shown to accumulate in P bodies (40), to inhibit translation and promote P-body formation when overexpressed (15), and to be required for the recruitment of Lsm1 to P bodies (42). To identify the regions of Pat1 that affected these three events, we examined how the different deletion constructs affected Pat1 accumulation in P bodies, Lsm1 recruitment to P bodies, and translation repression and P-body formation when overexpressed.
The region at residues 422 to 763 is required for Lsm1-GFP to accumulate in P bodies.
Previous work has shown that the ability of Lsm1-green fluorescent protein (GFP) to accumulate in P bodies is dependent on the presence of Pat1 (34). To determine what region(s) of Pat1 was required for Lsm1 recruitment to P bodies, we examined the presence of an Lsm1-GFP fusion protein in cells at high cell densities where P bodies are large and easily visualized (43). A formal caveat of these experiments is that we are examining a GFP-tagged version of Lsm1. However, since Lsm1-GFP was previously shown to require Pat1 function for localization to P bodies (41), and untagged Lsm1 is found in mammalian P bodies (25), this is a valid approach to studying Lsm1-GFP localization. As shown in Fig. 4, the distinct foci of Lsm1-GFP seen in a wild-type PAT1 background were no longer evident in the pat1 strain. Moreover, any Pat1 construct that lacked the region at residues 422 to 763 was deficient in recruiting Lsm1 to P bodies (constructs 10-763 through 697-763) (Fig. 4), including a precise deletion of this region (422-763). Note that both the region between residues 422 and 697 and the region between residues 697 and 763 are required for the recruitment of Lsm1 to P bodies by Pat1. In contrast, the regions at residues 10 to 254 and 254 to 422 are dispensable for the recruitment of Lsm1 to P bodies (constructs 10-254 and 254-422). This defines the region at residues 422 to 763 as being required for the recruitment of Lsm1, and presumably the entire Lsm1-7 complex, to P bodies.
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10-422 construct was effectively able to form foci of Lsm1-GFP, indicating that residues 422 to 763 are both necessary and sufficient (along with the N- and C-terminal amino acids) for the recruitment of Lsm1-GFP to P bodies. The region at residues 422 to 763 is required for Pat1-GFP to accumulate in P bodies. The data described above suggest that the region at residues 422 to 763 of Pat1 is required for the recruitment of Lsm1 to P bodies. The simplest explanation for this observation is that this region of Pat1 is required for Pat1 itself to accumulate in P bodies. To test this possibility, we fused a C-terminal GFP to Pat1 deletion variants lacking either residues 10 to 254 (a region that appeared to have no function yet), residues 254 to 422 (which were required for Pat1 to promote decapping), or residues 422 to 763 (which were required for the recruitment of Lsm1 to P bodies). We observed that residues 10 to 254 and 254 to 422 were not required for Pat1 to accumulate in P bodies (Fig. 5). In contrast, we observed that the region between residues 422 and 763 was required for Pat1 to localize to P bodies. This defines residues 422 to 763 as being required to target Pat1 to P bodies. Moreover, this suggests that residues 422 to 763 are required for Pat1 to recruit Lsm1, at least in part, by first localizing Pat1 to P bodies.
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Pat1 function in P-body assembly is upstream of decapping.
The data described above suggest a sequential model for Pat1 function. In the first step, the region of Pat1 between amino acids 422 and 763 first inhibits translation, increases the assembly of mRNPs that can accumulate in P bodies, and correspondingly has some effect on decapping efficiency. In this model, a second step in decapping would be promoted by the region between amino acids 254 and 422, which is enhanced by the translation repression/assembly function of residues 422 to 763. A prediction of this model is that different lesions in Pat1 should affect the accumulation of P bodies in different manners in a mid-log-phase culture, where yeast P bodies are typically small (40). Specifically, we predict that pat1 variants lacking amino acids 422 to 763 should lead to reduced P bodies compared to wild-type cells due to a defect in accumulation, whereas pat1 variants lacking just the region at residues 254 to 422 should show greatly increased levels of P bodies. Moreover, a larger deletion removing both regions would be expected have reduced levels of P bodies. To test these predictions, we examined the accumulation of Dcp2-GFP in various pat1 strains during mid-log-phase growth, where P bodies are small and increases in P-body size and number are easy to observe (41).
Consistent with the predictions described above, we observed that strains expressing 422-763 showed a reduction in the number of P-body-containing cells compared to the strains expressing the full-length Pat1, supporting the claim that the region at residues 422 to 763 affects P-body assembly (Fig. 7A and B). In contrast, strains expressing 254-422 showed an increase in the number and size of P bodies compared to the strains expressing full-length Pat1 (Fig. 7A and B). This is consistent with the region at residues 254 to 422 being required only for a late step in decapping after the formation of an mRNP that can accumulate in P bodies. Moreover, strains expressing Pat1 variants lacking both the region at residues 254 to 422 and the region at residues 422 to 763 (10-763 and 254-763) showed levels of P bodies similar to that of the 422-763 construct alone. This indicates that the accumulation of P bodies seen in the 254-422 strain is dependent on the ability of Pat1 to promote P-body assembly.
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Two-hybrid interactions reveal Pat1 interactions with Dcp1, Lsm1, and Edc3. To identify candidates for proteins that directly interact with Pat1, we screened for strong interactions between Pat1 and other components of P bodies by two-hybrid analysis. We observed that Pat1 showed strong two-hybrid interactions with Lsm1, Dcp1, and Edc3 (Fig. 8A). Following the initial identification of proteins that interact with Pat1, we used the Pat1 deletions to map the specific regions of Pat1 in which the interactions occur. It was determined that the Lsm1-Pat1 two-hybrid interaction requires amino acids 254 to 422 of Pat1, while the Pat1-Dcp1 and Pat1-Edc3 two-hybrid interactions required amino acids 422 to 797 (Fig. 8A).
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Pat1 is an RNA binding protein. The role of Pat1 in translation repression and mRNA decapping suggested that Pat1 might be an RNA binding protein. Moreover, the Xenopus ortholog of Pat1, P100, is capable of binding to DNA, although RNA binding was not tested in those experiments (37). In order to directly test if yeast Pat1 binds to RNA, we used RNA homopolymers bound to Sepharose beads as a means of assessing Pat1 binding to RNA. Pat1 was translated in the presence of [35S]methionine in an in vitro transcription and translation reaction. The lysate from this reaction was then incubated in the presence of poly(U)-bound Sepharose beads. As shown in Fig. 9, full-length Pat1 efficiently bound to poly(U) homopolymers in the presence of yeast tRNA as a nonspecific competitor (lane 2). This binding was successfully outcompeted in the presence of specific poly(U) competitor (Fig. 9, lanes 3 and 4) but not in the presence of poly(C) (lane 5). This binding was also specific to the poly(U) homopolymer, since no binding occurred when lysate was added to non-homopolymer-containing Sepharose, protein G-Sepharose (Fig. 9, lane 6). This observation indicates that Pat1 can bind RNA and shows a preference for poly(U) substrates.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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strains show a strong defect in mRNA decapping (7, 8, 21, 44) and have defects in translational repression during glucose deprivation (15, 23). Our results indicate that Pat1 plays two separable roles in the process of translation repression and mRNA decapping. In addition, we provide evidence that Pat1 binds RNA and has numerous interactions with mRNA decapping factors. As discussed in detail below, our results suggest a preliminary and initial model of Pat1 function wherein Pat1 first interacts with translating mRNAs through one or more domain to contribute to translation repression and the assembly of a P-body mRNP, followed by a second domain of Pat1 functioning to increase the actual rate of decapping following the formation of the P-body mRNP (Fig. 10).
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One unresolved issue is why residues 422 to 763 are sufficient to recruit Lsm1, and presumably the entire Lsm1-7 complex, to P bodies when only residues 254 to 422 showed interactions with Lsm1. We consider two possible explanations. First, it could be that there are additional interactions between the Lsm1-7 complex and Pat1 that we have not detected in the two-hybrid assay. Second, it could be that Pat1 binding to the P-body complex induces conformational changes in other proteins that then promote Lsm1-7 interactions with those components. Future experiments should be able to distinguish these possibilities.
The domain at residues 254 to 422 of Pat1 promotes mRNA decapping after P-body assembly.
Several observations identify Pat1 residues 254 to 422 as functioning to promote mRNA decapping after translation repression and the assembly of a P-body mRNP. The critical observation is that any Pat1 deletion construct lacking the domain at residues 254 to 422 is as defective in mRNA decapping as a pat1 strain (Fig. 3). In contrast, Pat1 deletion variants lacking the region at residues 254 to 422 are still able to assemble into P bodies (Fig. 5), recruit Lsm1-GFP and Dcp2-GFP to P bodies, and inhibit growth when overexpressed (Fig. 4, 6, and 7). Moreover, strains expressing Pat1 lacking the region at residues 254 to 422 accumulate large P bodies during mid-log-phase growth, consistent with a block to decapping (Fig. 7) Taken together, these observations suggest that the domain at residues 254 to 422 functions after the assembly of a translationally repressed mRNP that can accumulate in P bodies. Consistent with that view, the domain at residues 254 to 422 binds to Lsm1-7, which is known to affect decapping at a late stage after the assembly of an mRNP that can accumulate in P bodies (42).
An unresolved issue is the molecular event that the domain at residues 254 to 422 facilitates to promote decapping. One possibility is that additional interactions between the mRNA and/or additional decapping factors with this region of Pat1 stabilize the P-body mRNP and that this enhanced stability provides prolonged times for decapping. However, this model seems unlikely since we observed that the deletion of this region increases P-body formation (Fig. 7) and promotes even stronger translation repression when overexpressed (Fig. 6), which is consistent with the 254-422 construct leading to a block to decapping after the assembly of the P-body mRNP. Given this finding, a more likely possibility is that the domain at residues 254 to 422 functions to promote a conformational change in the P-body mRNP that activates the decapping enzyme (either by directly facilitating the formation of a more active conformation or by removing some inhibitory interactions).
Our results suggest that the function of the domain at residues 422 to 763 influences the ability of the domain at residues 254 to 422 to promote decapping. The key observation is that Pat1 variants lacking any part of the domain at residues 422 to 763 show a reduction in decapping efficiency (Fig. 3). Moreover, the residual decapping seen in the 422-763 variants is dependent on the region at residues 254 to 422 since variants lacking both residues 254 to 422 and 422 to 763 show no function of Pat1 in decapping (Fig. 3). These results are consistent with Pat1 functioning to first promote translation repression and the assembly of a P-body mRNP committed to decapping within which the region at residues 254 to 422 of Pat1 could function to enhance the actual rate of catalysis for decapping.
Pat1 is an RNA binding protein with two independent RNA binding domains. In this work, we also provide evidence that Pat1 is an RNA binding protein. The critical observation is that in vitro-synthesized Pat1 binds to poly(U)-Sepharose (Fig. 9). Moreover, this binding can be competed by poly(U) but not poly(C), thus suggesting that Pat1 prefers to bind poly(U)-rich RNAs. The ability of Pat1 to bind RNA is consistent with its coimmunoprecipitation with mRNA (45). In addition, it seems likely that this property will be conserved since the Xenopus ortholog of Pat1, P100, is capable of binding to DNA, although RNA was not tested in those experiments (37). Finally, because domains at both residues 254 to 422 and 422 to 763 of Pat1 are sufficient by themselves to bind RNA, Pat1 has two regions that can independently bind RNA. Interestingly, the two domains of Pat1 that bind RNA show no resemblance to known RNA binding domains, suggesting that these regions may represent novel RNA binding structures. An important area of future work will be to identify the regions of RNA binding with Pat1 in more detail and determine their preferred binding sequences and functional significances.
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ACKNOWLEDGMENTS |
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An NIH grant (R37 GM45443) and funds from the Howard Hughes Medical Institute supported this work.
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FOOTNOTES |
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Published ahead of print on 17 December 2007.
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