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Response to “Role of RPB7 in RNA pol I transcription in Trypanosoma brucei”
Molecular & Biochemical Parasitology 180 (2011) 45–46
Contents lists available at ScienceDirect
Molecular & Biochemical Parasitology
Letter to the Editors
Response to “Role of RPB7 in RNA pol I transcription in Trypanosoma brucei” In their letter, Navarro et al. [1] criticize our approaches and conclusion that RPB7 is not required for RNA polymerase (pol) I transcription in Trypanosoma brucei [2]. Our results are in sharp contrast to their previously published work which claimed that RPB7 is essential for RNA pol I transcription [3]. We used several independent criteria to evaluate the functional role of RPB7 in RNA pol I transcription, namely reciprocal co-immunoprecipitation (co-IP), tandem affinity purification (TAP), chromatin immunoprecipitation (ChIP), in vitro transcription assays and indirect immunofluorescence (IF) microscopy. In no case did we find evidence that RPB7 has an RNA pol II-independent function in RNA pol I transcription or that this protein is stably associated with RNA pol I. We had hoped that the letter by Navarro et al. would help to resolve the discrepancy between our two studies; instead we had to read some rather far-fetched arguments why our results could be artifacts. Navarro et al. describe RPB7 to be essential for RNA pol I transcription and, at the same time, not to be a bona fide RNA pol subunit [1]. Conversely, though dissociable after transcription initiation [4], RPB7 has been described as an essential RNA pol II subunit that is required for promoter-dependent transcription initiation [5] and ChIP assays demonstrated that RPB7 occupancy is congruent to that of the non-dissociable subunit RPB3 [6]. Accordingly, we readily found T. brucei RPB7 as part of RNA pol II complexes and crosslinked to RNA pol II transcription units while no such link to RNA pol I was detectable [2]. Navarro et al. also state that they have “previously described a low-affinity interaction of RPB7 with RPA1” [1]. The term “low affinity”, however, was not used in their original publication [3] and their interpretation of low affinity remains unclear. The most obvious discrepancy between the two studies resides in the in vitro transcription outcomes. In our assays, RPB7 depletion of extract nearly abolished RNA pol II transcription but left RNA pol I transcription unaffected [2] whereas a similar approach by Penate et al. interfered with RNA pol I transcription [3]. It should be noted that our system allows co-transcription of RNA pol I and II promoter templates and, due to primer extension-derived transcription signals, monitors accurate transcription initiation. In comparison, the transcription signals provided by Penate et al. [3] depended on a Gless cassette that was introduced downstream of the transcription initiation site. Hence, there is no discrimination between accurately initiated and non-specific read-through transcription. In addition, individual reactions were not internally controlled. This is particularly bothersome because their analysis was not quantitatively
DOIs of original articles: 10.1016/j.molbiopara.2011.07.005
10.1016/j.molbiopara.2011.06.008,
0166-6851/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2011.07.006
assessed, e.g. based on signal quantification of repeated experiments. According to Navarro et al. the lack of an effect on RNA pol I transcription in our assays could stem from residual amounts of RPB7 left in the extract [1]. Since our extracts were prepared from cells that exclusively expressed the essential RPB7 as a TAP tag fusion, this would mean that the tandem protein A domains of tagged RPB7 were masked in the RNA pol I complex but accessible in the RNA pol II complex. Having precipitated and purified more than 30 different TAP-tagged proteins (see for example [7–9]), we have not come across a single case in which the large tandem protein A domains of the TAP tag were inaccessible to IgG beads or antibodies. Navarro et al. stated that in our co-IP analysis 25% of RPB7 interacted with RPB1 and only 1% of RPB7 interacted with RPA1 [1]. This is not true. In fact, we did not find any interaction between RPB7 and the RNA pol I-specific subunit RPB6z in both reciprocal co-IP and RPB7 TAP whereas in both assays RPB7 was stably associated with RNA pol II. We did, however, acknowledge that, due to the ambiguity of quantifying large amounts of immunoprecipitate, we could not exclude the possibility that a very small amount of RPB7 (<1%) was associated with RPB6z. In regard to the amount of active RNA pol I in extracts, a study in yeast found that up to 98% of RNA pol I was inactive. However, the inactivity was due to the dissociation of the transcription factor RRN3 from RNA pol I and not to the lack of an RNA pol I subunit, e.g. the RNA pol I paralog of RPB7 termed RPA43 [10]. Navarro et al. criticize our ChIP approach because we, like many others, compare the enrichment of precipitated DNA over a negative control immunoprecipitation in which we use a comparable, non-specific immune serum. While the suggested normalization to input amount is valid, too, this kind of analysis promotes the usage of extremely low ratios of chromatin input and antibody beads. More importantly, our ChIP analysis demonstrated that identically tagged RPB7, RPB6z and RPB9 could be effectively and specifically cross-linked to chromatin. In all cases, we found congruency of RPB7 and RPB9 (RNA pol II) occupancies that did not correlate with RPB6z (RNA pol I) occupancy. In this regard, we find it inappropriate that Navarro et al. mention unpublished data in their support which cannot be evaluated yet. Concerning IF microscopy, we maintain that the co-localization mask provided by Penate et al. [3] cannot discriminate between adjacent signals and true co-localization. Since they concluded that RPB7 is generally required for RNA pol I including transcription of ribosomal RNA genes, our approach to localize RPB7-PTP in nucleoli of transiently transfected procyclics is valid. Importantly, a major difference between our study and that of Penate et al. was that we used functional protein tags (exclusive expression of essential, tagged proteins) and they used a polyclonal anti-RPB7 antiserum. Since it is very likely that trypanosome RNA pol I requires an RPA43 ortholog [11] representing an RPB7 para-
46
Letter to the Editors / Molecular & Biochemical Parasitology 180 (2011) 45–46
log, Navarro et al. should make sure that their antibody does not cross-react with an RNA pol I-specific protein, e.g. TbRPA31 [12], the protein we favor in retrospect to be the RNA pol I paralog of RPB7 (see conclusion section in [2]). Finally, we are convinced that our data, in contrast to the titlebased conclusion of Penate et al. [3], demonstrate that RPB7 has no essential role in T. brucei RNA pol I transcription; it does not replace the function of an RPA43 ortholog. As we state in our publication, we cannot rule out a post-transcriptional or RNA pol II-based role of RPB7 in the expression of RNA pol I-synthesized transcripts because this was not the aim of our analysis. References [1] Navarro M, Penate X, Landeira D, et al. Role of RPB7 in RNA pol I transcription in Trypanosoma brucei. Mol Biochem Parasitol 2011, doi:10.1016/j.molbiopara.2011.06.005. [2] Park SH, Nguyen TN, Kirkham JK, et al. Transcription by the multifunctional RNA polymerase I in Trypanosoma brucei functions independently of RPB7. Mol Biochem Parasitol 2011;180:35–42. [3] Penate X, Lopez-Farfan D, Landeira D, et al. RNA pol II subunit RPB7 is required for RNA pol I-mediated transcription in Trypanosoma brucei. EMBO Rep 2009;10:252–7. [4] Harel-Sharvit L, Eldad N, Haimovich G, et al. RNA polymerase II subunits link transcription and mRNA decay to translation. Cell 2010;143:552–63. [5] Edwards AM, Kane CM, Young RA, et al. Two dissociable subunits of yeast RNA polymerase II stimulate the initiation of transcription at a promoter in vitro. J Biol Chem 1991;266:71–5. [6] Jasiak AJ, Hartmann H, Karakasili E, et al. Genome-associated RNA polymerase II includes the dissociable Rpb4/7 subcomplex. J Biol Chem 2008;283:26423–7. [7] Brandenburg J, Schimanski B, Nogoceke E, et al. Multifunctional class I transcription in Trypanosoma brucei depends on a novel protein complex. EMBO J 2007;26:4856–66.
[8] Lee JH, Jung HS, Günzl A. Transcriptionally active TFIIH of the early-diverged eukaryote Trypanosoma brucei harbors two novel core subunits but not a cyclinactivating kinase complex. Nucleic Acids Res 2009;37:3811–20. [9] Lee JH, Cai G, Panigrahi AK, et al. A TFIIH-associated mediator head is a basal factor of small nuclear spliced leader RNA gene transcription in early-diverged trypanosomes. Mol Cell Biol 2010;30:5502–13. [10] Milkereit P, Tschochner H. A specialized form of RNA polymerase I, essential for initiation and growth-dependent regulation of rRNA synthesis, is disrupted during transcription. EMBO J 1998;17:3692–703. [11] Kuhn CD, Geiger SR, Baumli S, et al. Functional architecture of RNA polymerase I. Cell 2007;131:1260–72. [12] Nguyen TN, Schimanski B, Günzl A. Active RNA polymerase I of Trypanosoma brucei harbors a novel subunit essential for transcription. Mol Cell Biol 2007;27:6254–63.
Arthur Günzl ∗ Sung Hee Park Tu N. Nguyen Justin K. Kirkham Ju Huck Lee Department of Genetics and Developmental Biology, University of Connecticut Health Center, 400 Farmington Avenue, Farmington, CT 06030-6403, USA ∗ Corresponding
author. Tel.: +1 860 679 8878; fax: +1 860 679 8345. E-mail address: [email protected] (A. Günzl) 30 June 2011 11 July 2011 Available online 23 July 2011
Contents lists available at ScienceDirect
Molecular & Biochemical Parasitology
Letter to the Editors
Response to “Role of RPB7 in RNA pol I transcription in Trypanosoma brucei” In their letter, Navarro et al. [1] criticize our approaches and conclusion that RPB7 is not required for RNA polymerase (pol) I transcription in Trypanosoma brucei [2]. Our results are in sharp contrast to their previously published work which claimed that RPB7 is essential for RNA pol I transcription [3]. We used several independent criteria to evaluate the functional role of RPB7 in RNA pol I transcription, namely reciprocal co-immunoprecipitation (co-IP), tandem affinity purification (TAP), chromatin immunoprecipitation (ChIP), in vitro transcription assays and indirect immunofluorescence (IF) microscopy. In no case did we find evidence that RPB7 has an RNA pol II-independent function in RNA pol I transcription or that this protein is stably associated with RNA pol I. We had hoped that the letter by Navarro et al. would help to resolve the discrepancy between our two studies; instead we had to read some rather far-fetched arguments why our results could be artifacts. Navarro et al. describe RPB7 to be essential for RNA pol I transcription and, at the same time, not to be a bona fide RNA pol subunit [1]. Conversely, though dissociable after transcription initiation [4], RPB7 has been described as an essential RNA pol II subunit that is required for promoter-dependent transcription initiation [5] and ChIP assays demonstrated that RPB7 occupancy is congruent to that of the non-dissociable subunit RPB3 [6]. Accordingly, we readily found T. brucei RPB7 as part of RNA pol II complexes and crosslinked to RNA pol II transcription units while no such link to RNA pol I was detectable [2]. Navarro et al. also state that they have “previously described a low-affinity interaction of RPB7 with RPA1” [1]. The term “low affinity”, however, was not used in their original publication [3] and their interpretation of low affinity remains unclear. The most obvious discrepancy between the two studies resides in the in vitro transcription outcomes. In our assays, RPB7 depletion of extract nearly abolished RNA pol II transcription but left RNA pol I transcription unaffected [2] whereas a similar approach by Penate et al. interfered with RNA pol I transcription [3]. It should be noted that our system allows co-transcription of RNA pol I and II promoter templates and, due to primer extension-derived transcription signals, monitors accurate transcription initiation. In comparison, the transcription signals provided by Penate et al. [3] depended on a Gless cassette that was introduced downstream of the transcription initiation site. Hence, there is no discrimination between accurately initiated and non-specific read-through transcription. In addition, individual reactions were not internally controlled. This is particularly bothersome because their analysis was not quantitatively
DOIs of original articles: 10.1016/j.molbiopara.2011.07.005
10.1016/j.molbiopara.2011.06.008,
0166-6851/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2011.07.006
assessed, e.g. based on signal quantification of repeated experiments. According to Navarro et al. the lack of an effect on RNA pol I transcription in our assays could stem from residual amounts of RPB7 left in the extract [1]. Since our extracts were prepared from cells that exclusively expressed the essential RPB7 as a TAP tag fusion, this would mean that the tandem protein A domains of tagged RPB7 were masked in the RNA pol I complex but accessible in the RNA pol II complex. Having precipitated and purified more than 30 different TAP-tagged proteins (see for example [7–9]), we have not come across a single case in which the large tandem protein A domains of the TAP tag were inaccessible to IgG beads or antibodies. Navarro et al. stated that in our co-IP analysis 25% of RPB7 interacted with RPB1 and only 1% of RPB7 interacted with RPA1 [1]. This is not true. In fact, we did not find any interaction between RPB7 and the RNA pol I-specific subunit RPB6z in both reciprocal co-IP and RPB7 TAP whereas in both assays RPB7 was stably associated with RNA pol II. We did, however, acknowledge that, due to the ambiguity of quantifying large amounts of immunoprecipitate, we could not exclude the possibility that a very small amount of RPB7 (<1%) was associated with RPB6z. In regard to the amount of active RNA pol I in extracts, a study in yeast found that up to 98% of RNA pol I was inactive. However, the inactivity was due to the dissociation of the transcription factor RRN3 from RNA pol I and not to the lack of an RNA pol I subunit, e.g. the RNA pol I paralog of RPB7 termed RPA43 [10]. Navarro et al. criticize our ChIP approach because we, like many others, compare the enrichment of precipitated DNA over a negative control immunoprecipitation in which we use a comparable, non-specific immune serum. While the suggested normalization to input amount is valid, too, this kind of analysis promotes the usage of extremely low ratios of chromatin input and antibody beads. More importantly, our ChIP analysis demonstrated that identically tagged RPB7, RPB6z and RPB9 could be effectively and specifically cross-linked to chromatin. In all cases, we found congruency of RPB7 and RPB9 (RNA pol II) occupancies that did not correlate with RPB6z (RNA pol I) occupancy. In this regard, we find it inappropriate that Navarro et al. mention unpublished data in their support which cannot be evaluated yet. Concerning IF microscopy, we maintain that the co-localization mask provided by Penate et al. [3] cannot discriminate between adjacent signals and true co-localization. Since they concluded that RPB7 is generally required for RNA pol I including transcription of ribosomal RNA genes, our approach to localize RPB7-PTP in nucleoli of transiently transfected procyclics is valid. Importantly, a major difference between our study and that of Penate et al. was that we used functional protein tags (exclusive expression of essential, tagged proteins) and they used a polyclonal anti-RPB7 antiserum. Since it is very likely that trypanosome RNA pol I requires an RPA43 ortholog [11] representing an RPB7 para-
46
Letter to the Editors / Molecular & Biochemical Parasitology 180 (2011) 45–46
log, Navarro et al. should make sure that their antibody does not cross-react with an RNA pol I-specific protein, e.g. TbRPA31 [12], the protein we favor in retrospect to be the RNA pol I paralog of RPB7 (see conclusion section in [2]). Finally, we are convinced that our data, in contrast to the titlebased conclusion of Penate et al. [3], demonstrate that RPB7 has no essential role in T. brucei RNA pol I transcription; it does not replace the function of an RPA43 ortholog. As we state in our publication, we cannot rule out a post-transcriptional or RNA pol II-based role of RPB7 in the expression of RNA pol I-synthesized transcripts because this was not the aim of our analysis. References [1] Navarro M, Penate X, Landeira D, et al. Role of RPB7 in RNA pol I transcription in Trypanosoma brucei. Mol Biochem Parasitol 2011, doi:10.1016/j.molbiopara.2011.06.005. [2] Park SH, Nguyen TN, Kirkham JK, et al. Transcription by the multifunctional RNA polymerase I in Trypanosoma brucei functions independently of RPB7. Mol Biochem Parasitol 2011;180:35–42. [3] Penate X, Lopez-Farfan D, Landeira D, et al. RNA pol II subunit RPB7 is required for RNA pol I-mediated transcription in Trypanosoma brucei. EMBO Rep 2009;10:252–7. [4] Harel-Sharvit L, Eldad N, Haimovich G, et al. RNA polymerase II subunits link transcription and mRNA decay to translation. Cell 2010;143:552–63. [5] Edwards AM, Kane CM, Young RA, et al. Two dissociable subunits of yeast RNA polymerase II stimulate the initiation of transcription at a promoter in vitro. J Biol Chem 1991;266:71–5. [6] Jasiak AJ, Hartmann H, Karakasili E, et al. Genome-associated RNA polymerase II includes the dissociable Rpb4/7 subcomplex. J Biol Chem 2008;283:26423–7. [7] Brandenburg J, Schimanski B, Nogoceke E, et al. Multifunctional class I transcription in Trypanosoma brucei depends on a novel protein complex. EMBO J 2007;26:4856–66.
[8] Lee JH, Jung HS, Günzl A. Transcriptionally active TFIIH of the early-diverged eukaryote Trypanosoma brucei harbors two novel core subunits but not a cyclinactivating kinase complex. Nucleic Acids Res 2009;37:3811–20. [9] Lee JH, Cai G, Panigrahi AK, et al. A TFIIH-associated mediator head is a basal factor of small nuclear spliced leader RNA gene transcription in early-diverged trypanosomes. Mol Cell Biol 2010;30:5502–13. [10] Milkereit P, Tschochner H. A specialized form of RNA polymerase I, essential for initiation and growth-dependent regulation of rRNA synthesis, is disrupted during transcription. EMBO J 1998;17:3692–703. [11] Kuhn CD, Geiger SR, Baumli S, et al. Functional architecture of RNA polymerase I. Cell 2007;131:1260–72. [12] Nguyen TN, Schimanski B, Günzl A. Active RNA polymerase I of Trypanosoma brucei harbors a novel subunit essential for transcription. Mol Cell Biol 2007;27:6254–63.
Arthur Günzl ∗ Sung Hee Park Tu N. Nguyen Justin K. Kirkham Ju Huck Lee Department of Genetics and Developmental Biology, University of Connecticut Health Center, 400 Farmington Avenue, Farmington, CT 06030-6403, USA ∗ Corresponding
author. Tel.: +1 860 679 8878; fax: +1 860 679 8345. E-mail address: [email protected] (A. Günzl) 30 June 2011 11 July 2011 Available online 23 July 2011