Rap1 interacts with Fhl1 and with Ifh1.
Although ChIP experiments clearly demonstrate that Fhl1 is associated with RP gene promoters, we have been unable to detect a direct interaction of Fhl1 with DNA of the RP gene promoter, as measured by gel shift in vitro. Furthermore, deletion of the putative DNA binding domain of Fhl1 does not cause a significant growth defect (33
). These results prompted us to ask if Fhl1 is brought to RP genes, not by its FH domain, but by interacting with Rap1. Indeed, Co-IP analysis using anti-Rap1 antibody shows that Rap1 can associate not only with Fhl1 but with Ifh1 as well (Fig. , left). The interaction is unaffected by the presence of ethidium bromide, known to dissociate proteins from DNA (21
) (Fig. , right), suggesting that Rap1 can associate with Fhl1 and Ifh1 in a DNA-independent manner. The FHA domain, found in many “forkhead”-type proteins, is known to bind to phosphopeptides (6
) and is essential for Fhl1 function and for its interaction with Ifh1. Even a point mutation in the FHA domain (S325R) has a serious impact on its function (33
). Interestingly, mutation of neither the FH nor the FHA domains of Fhl1 affects the Co-IP of either Fhl1 or Ifh1 by Rap1 (Fig. , lanes 7, 8, and 9). This observation leads to the unexpected conclusion that Ifh1 can interact with Rap1 independently of Fhl1.
FIG. 1. Rap1 interacts with both Fhl1 and Ifh1. (A) Co-IP was carried out using rabbit polyclonal anti-Rap1 (αRap1) antibody with extracts prepared from strain DR36 (FHL1-HA3 IFH1-Myc9 double tagged). The immunoprecipitated protein complex was resuspended (more ...) Ifh1 is found as a high-MW complex.
To clarify the relationship between Rap1, Fhl1, and Ifh1, we asked whether these three proteins could be found in a stable complex. Glycerol gradient analysis of an extract of wild-type cells is shown in Fig. . Strikingly, Ifh1 sediments as a complex (or complexes) with an apparent molecular mass of about 350 to 400 kDa, significantly larger than expected for a 122-kDa protein. Both Rap1 and Fhl1 sediment primarily as free proteins near the top of the gradient but with a pronounced tail down the gradient. (The significance of the small amount of Rap1 that migrates slightly faster in the gel than most of the Rap1, in fractions 13 and 15, is under investigation.) Since some of the Rap1 and Fhl1 cosediments with Ifh1 in fractions 9 and 11, we asked if they were associated. In the higher-molecular-weight (MW) gradient fractions, not only does Ifh1 coimmunoprecipitate Fhl1, but Rap1 can also coimmunoprecipitate both Fhl1 and Ifh1 (Fig. ). The efficiency with which Rap1 can coimmunoprecipitate Fhl1 is much greater in the high-MW fractions (fractions 9 and 11) than at the top of the gradient (fractions 3 and 5). Thus, in these fractions Rap1 and Fhl1 are complexed with at least a part of Ifh1.
FIG. 2. Ifh1 is found as a high-MW complex. (A) Extract prepared from strain DR36 (FHL1-HA3 IFH1-Myc9) was loaded onto a 10 to 30% glycerol gradient and centrifuged for 5 h at 49 krpm using an SW50.1 rotor. Two hundred-microliter fractions were collected. (more ...) Ifh1 is in a complex with rRNA processing factors.
Extracts of cells carrying Fhl1-TAP, Ifh1-TAP, and TAP-Rap1 (the latter tagged at the N terminus [see Materials and Methods] because a C-terminal tag interferes with function) (Fig. ) were used to estimate the relative amounts of the three proteins using a slot blot (Fig. ) The results suggest that Fhl1 and Ifh1 are approximately equimolar but only 10 to 20% the level of Rap1, in confirmation of the genomewide analysis (12
), which is not surprising because Rap1 is found at many sites in the genome. However, since any complex with Fhl1 could account for only a small fraction of the Ifh1, it seemed likely that most of the Ifh1 was associated with something else. To identify other proteins that are complexed with Ifh1, we carried a TAP-tagged Ifh1 through the first step of purification (31
), followed by a glycerol gradient, with the hope that most of the proteins that are associated with Ifh1 in fractions 9 to 11 can be detected by silver staining. As is apparent from Fig. , several proteins copurified with Ifh1 on the gradient (fractions 9 and 11). The most prominent of these was identified by mass spectrometry as Utp22, at 140 kDa, consistent with its migration in the denaturing gel. Note that Ifh1, also identified by mass spectrometry, migrates anomalously slowly. In addition, the lower bands contained Rrp7 and the α subunit of CK2. These results are consistent with a recent analysis of TAP-tagged Utp22 in a quite different strain (31
) that identified a complex termed UTP-C. This complex contained Rrp7, the four subunits of CK2, and Ifh1 (see supplemental data in reference 19
). However, Ifh1 was not visible on the accompanying gel (see Fig. of reference 19
), presumably because we find it to be highly labile to proteolytic digestion. Utp22 has also been identified as part of a much larger, 2.2-MDa, complex of proteins associated with U3 snoRNA (the “SSU processome”) and implicated in the early cleavage steps of 35S pre-rRNA (3
). Rrp7 has been implicated in a later step in the assembly of 40S ribosomal subunits (2
). The absence of either Utp22 or Rrp7 leads to a deficiency in the formation of 40S ribosomal subunits.
FIG. 3. Ifh1 is in a complex with rRNA processing factors. (A) Western blot analysis performed on whole-cell extracts prepared from the indicated wild-type or TAP-tagged strains, each under its own promoter (see Materials and Methods). HRP-conjugated chicken (more ...)
To validate the above results and to determine the glycerol gradient pattern of Utp22, Rrp7, and the β subunit of the CK2 protein (Ckb2), we C-terminally FLAG tagged Rrp7 and Ckb2 and TAP tagged Utp22 in a strain carrying Fhl1-HA and Ifh1-Myc (strains DR114, DR115, and DR113, respectively [see Table ]). A summary of the glycerol gradient patterns from the three strains is shown in Fig. . Both Rrp7-FLAG and Utp22-TAP peak at fractions 9, 11, and 13. A considerable portion of the Ckb2-FLAG protein is also detected in those fractions. It is noteworthy that substantial fractions of Utp22 and Rrp7 are present in the pellet, consistent with the finding that Utp22 is a member of a large nucleolar U3 ribonucleoprotein complex (3
) and that both Utp22 and Rrp7 have been identified in a 90S preribosome particle (8
). In order to demonstrate that the complex with Ifh1 was distinct from the U3-containing processome, we carried out Co-IP analysis of an extract of strain JD001, carrying Ifh1-Myc9 and Utp22-FLAG. As is apparent from Fig. , Ifh-Myc can coimmunoprecipitate Utp22-FLAG but not U3 RNA (lane 3), while Utp22-FLAG can coimmunoprecipitate both Ifh1 and U3 RNA (lane 4). The results shown in Fig. strongly suggest that much of the Ifh1 in fractions 9 to 13 of the glycerol gradient is present in a complex with CK2, Utp22, and Rrp7. We term this the CURI complex (C
FIG. 4. Portions of Ckb2, Rrp7, and Utp22 cosediment with Ifh1. (A) Glycerol gradient analysis was performed on extracts prepared from cultures of strains DR113, DR114, and DR115. Aliquots (15 μl) from the indicated fractions were analyzed by SDS-polyacrylamide (more ...) Fhl1 is loosely associated with the CURI complex.
Although the purified CURI complex in Fig. showed no silver-stained band corresponding to Fhl1, Western blotting revealed a minute amount of Fhl1 but no Rap1 (data not shown). This suggested that Fhl1 might be loosely associated with the CURI complex. To determine if this is the case, we performed Co-IP experiments on peak fractions containing the CURI complex with the respective FLAG-tagged or TAP-tagged proteins. As expected, Ifh1-Myc9 could be coimmunoprecipitated along with Utp22-TAP by IgG-Sepharose from a strain carrying Utp22-TAP strain (DR113) and not from a strain where Utp22 is untagged (Fig. ). Similarly, rabbit anti-FLAG antibody could efficiently coimmunoprecipitate Ifh1-Myc9 from strains DR114 (Rrp7-FLAG) and DR115 (Ckb2-FLAG) (Fig. ). Furthermore, in all of these cases, a small but reproducible amount of Fhl1 could be coimmunoprecipitated from these fractions with Utp22, Rrp7, and Ckb2 (Fig. , middle rows). Note that although the anti-FLAG antibody can immunoprecipitate the small amount of the FLAG-tagged Rrp7 or Ckb2 that is present in the fractions near the top of the gradient, it was unable to coimmunoprecipitate (Fig. ) any of the relatively large amount of Fhl1 from these fractions (Fig. ), suggesting that the association of Fhl1 with the CURI complex is authentic. From none of the fractions was Rap1 immunoprecipitated by Utp22, Rrp7, or Ckb2 (data not shown).
FIG. 5. The CURI complex interacts with Fhl1. (A) Co-IP using IgG-Sepharose was performed on a pool of fractions 9 and 11 of the glycerol gradients from extracts prepared from strains DR113 (UTP22-TAP) and DR47 (as a negative control). Western blotting was performed (more ...)
To confirm that Fhl1 indeed associates with the CURI complex within the cell, we performed Co-IP analysis on a whole-cell extract. As shown in Fig. , Fhl1-HA3 can coimmunoprecipitate Rrp7-FLAG (lane 5) or Ckb2-FLAG (lane 6). Conversely, Utp22-TAP or Ckb2-FLAG can coimmunoprecipitate Fhl1-HA3 (Fig. , lane 4, and 6C, lane 3, respectively). It is noteworthy that immunoprecipitation of Utp22 or Ckb2 does not coimmunoprecipitate Rap1 (Fig. ). Conversely, anti-Rap1 does not detectably coimmunoprecipitate Rrp7 (Fig. , lane 2) or Utp22 (Fig. , lane 5) under conditions where it does coimmunoprecipitate Fhl1 and Ifh1 (lane 2). Taken together, the results shown thus far suggest that (i) a small proportion of Fhl1 and Ifh1 is associated with Rap1, (ii) Ifh1 is mostly associated with the CURI complex, (iii) Fhl1 is weakly associated with the CURI complex, and (iv) Rap1 is not associated with the CURI complex.
FIG. 6. Fhl1 but not Rap1 interacts with components of the CURI complex in whole-cell extracts. (A) Co-IP was performed using anti-HA (α-HA) antibody or anti-mouse IgG (α-IgG) on strains DR114 (Rrp7-FLAG) and DR115 (Ckb2-FLAG), respectively. This (more ...) Fhl1 influences the stability of the CURI complex.
Cells in which the interaction of Ifh1 with Fhl1 is prevented, either by ablation of Fhl1 (16
) or by deletion of the FHA domain of Fhl1 (33
), are alive but grow exceedingly slowly. In light of these earlier findings and of the observation that Fhl1 interacts with the CURI complex, we wished to determine the fate of the CURI complex in the ΔFHL1
strains. As shown in Fig. , the deletion of Fhl1 (upper panel) or of the FHA domain (lower panel) has at least three effects on the pattern of migration (compare Fig. with Fig. ). First, a substantial amount of the Ifh1 now sediments more slowly, although a fraction of the Ifh1 still sediments at its previous location in fractions 9 to 13. Second, most of the Rrp7 sediments towards the top of the gradient. This is in marked contrast to the wild-type cells, where Rrp7 comigrates with Ifh1 in fractions 9 to 13. Third, Rap1 no longer streaks down the gradient. The altered sedimentation patterns of Ifh1 and of Rap1 are remarkably similar in the strain from which the FHA domain has been deleted from Fhl1 (Fig. , lower panel). Taken together, these results suggest that Fhl1 not only interacts with the CURI complex but also plays some role in maintaining the stability of the complex. On the other hand, we cannot rule out the possibility that these effects are secondary to the very slow growth and very low ribosome content of ΔFHL1
FIG. 7. Fhl1 influences the stability of the CURI complex. Glycerol gradient analysis was performed on extracts prepared from DR116, DR117, and DR118 strains. In each, FHL1 is deleted and Utp22, Rrp7, and Ckb2 are tagged as indicated. Aliquots (15 μl) (more ...) Ifh1 can be phosphorylated by CK2.
The presence of CK2 in the CURI complex is intriguing because CK2 has been implicated in the regulation of transcription by RNA polymerase I (Pol I) (34
), RNA Pol II (24
), and RNA Pol III (13
). To investigate whether CK2 phosphorylates members of the CURI complex, we incubated γ-[32
P]ATP with CURI-containing fractions extracted from strains YZ146 and DR23, carrying Ifh1-HA3
, respectively. The samples were immunoprecipitated with anti-HA antibody, and the result was displayed on two SDS gels (Fig. ): one subjected to autoradiography (upper) and the other probed with anti-HA antibody after Western blotting (lower). It is evident that Ifh1 is phosphorylated (lane 1), while Fhl1 is not (lane 5). To confirm that CK2 is the kinase responsible, we showed that heparin, a specific inhibitor of CK2 (15
), abolishes labeling of Ifh1 (lane 2). Furthermore, the kinase is just as effective using labeled GTP in place of ATP, another diagnostic for CK2 (28
) (Fig. , lanes 3 and 4). No phosphorylation of other members of CURI was detected in analogous experiments (data not shown).
FIG. 8. Phosphorylation of Ifh1 by CK2. Fractions of the glycerol gradient containing the CURI complex from strain YZ146 (Ifh1-HA3) were incubated with γ-32P-labeled ATP (lanes 1 and 2) or GTP (lanes 3 and 4), in the absence (lanes 1 and 3) or presence (more ...) CURI complex and transcription.
To ask if the CURI complex is involved in the activation of transcription of RP genes by Ifh1, we performed ChIP analysis with each of the components of the CURI complex. As seen in Fig. , the data show that while Ifh1 is found at the RP genes, the other three components, Ckb2, Rrp7, and Utp22, are not. Thus, Ifh1 seems to appear in two alternative forms: as either a member of the CURI complex or resident at the promoter of an RP gene, presumably in the company of Fhl1 and Rap1.
FIG. 9. Utp22, Rrp7, and Ckb2 are not present on the promoters of RP genes. ChIP was performed on strains harboring TAP-tagged IFH1 (DR14) and strains from Open Biosystems, Inc., carrying TAP-tagged versions of CKB2, RRP7, and UTP22 (see Materials and Methods.) (more ...) The CURI complex as coordinator of rRNA and RP production.
The presence of both Ifh1 and Utp22 and Rrp7 in the CURI complex led us to hypothesize that CURI might connect Pol I transcription of rRNA genes with Pol II transcription of RP genes. The simplest formulation is shown in Fig. . When rRNA transcription is active, much of the Utp22 and Rrp7 is tied up in processing the pre-rRNA, thus freeing Ifh1 to associate with Fhl1 and Rap1 to activate transcription of RP genes. Conversely, when rRNA transcription is reduced, Utp22 and Rrp7 are free to bind more Ifh1, thereby reducing transcription of RP genes.
FIG. 10. Model of CURI coupling rRNA and RP production. Ifh1 participates in (at least) two interactions. To the left is an RP promoter to which two molecules of Rap1 bind, simultaneously bending the DNA and clearing it of nucleosomes. Fhl1 and Ifh1 bind (not (more ...)
As a test of this hypothesis, we generated strains in which either UTP22
, and as a control, UTP7
, or UTP14
, were put under GAL control. In galactose medium, these strains grow as wild type. On being shifted to a noninducing, nonrepressive carbon source such as raffinose, growth slows substantially after 12 h and is limited for the protein in question. Analysis of the RNA from such strains under those conditions (Fig. ) shows that the level of 20S pre-rRNA is greatly reduced, as described previously for Rrp7 (2
) and for many of the Utp proteins (5
). For strains limited for Utp22 or Rrp7, the mRNA level of the five RP genes tested was substantially elevated (Fig. ), just as we would predict from the hypothesis described above. In contrast, the levels of RP mRNAs in strains limited for Utp7, -11, or -14 showed only a small increase, about 50%. While this result suggests that there may be some feedback from the deprivation of ribosomes, this 50% increase is far less than the three- to fourfold increase in the level of RP mRNAs when the cells are limited for members of the CURI complex, Utp22 or Rrp7. It is noteworthy that limiting either Utp22 or Rrp7 leads to an apparent decrease in the mRNA from ACT1
, as well as those from TEF1
, whose transcription is also dependent on Rap1.
FIG. 11. Depletion of Utp22 or Rrp7 leads to overexpression of RP mRNAs. Cells of the indicated genotype (where G-UTP22 means that transcription of UTP22 mRNA was under GAL control, etc.) were grown in YP-Gal, collected by filtration, and then grown in YP-raffinose (more ...)
An additional prediction of the model in Fig. is that restoring Rrp7 or Utp22 would then reduce the level of RP mRNA. As shown in Fig. , this is indeed the case. When galactose was added to restore Rrp7 or Utp22, the level of RP mRNA was reduced in spite of the fact that the growth rate rapidly increased. This experiment is entirely consistent with the model shown in Fig. .
The results in Fig. are quite remarkable for the following two reasons. (i) The mRNA level of RP genes is usually proportional to growth; in this case, it is the opposite. (ii) The mRNA level of RP genes in normal cells is already very high (17
). To increase that level by severalfold means that the RP mRNAs will be crowding out the other mRNAs of the cell. Indeed, the raw data of Fig. suggest that this is the case.