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Influences along the path to maturity: Regulation of cellular levels of RNA
Biochimica et Biophysica Acta 1759 (2006) 385 – 387 www.elsevier.com/locate/bbaexp
Review
Influences along the path to maturity: Regulation of cellular levels of RNA Michelle Craig Barton ⁎ Department of Biochemistry and Molecular Biology, Program in Genes and Development, Graduate School of Biomedical Sciences, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA Received 28 August 2006; accepted 29 August 2006 Available online 5 September 2006
Abstract Initiation of RNA transcription may be a rate-limiting step in gene expression but it is only the first of many regulatory processes that impinge on nascent RNA along its path to maturity. Discontinuity between gene expression patterns within the nucleus and the cytoplasm suggests that multiple post-transcription regulatory points greatly influence the final RNA product, even to the extent of dramatically shifting the gene targets identified as a defined regulatory response. © 2006 Elsevier B.V. All rights reserved. Keywords: Microarray; Dioxin; RNA; Transcription; Processing
In gene expression, the number of regulatory controls between signal input and functional outcome comprises a long and, seemingly, ever-expanding list. To initiate the process of understanding the regulatory impact of each control point, the simple task of measuring steady-state levels of RNA, encoded by a given gene, is generally used as a first estimate of regulatory response. Recent results reported by Tomlinson and colleagues [1], in this issue of Biochimica et Biophysica Acta, bring into question whether even this task is so simple. Two major points are made in this article: (1) Global expression patterns are established by numerous processes that act on nascent RNA to alter its sequence, its localization and even its existence. (2) The functional term “total RNA” requires more precise definition, as the population of RNAs present in each subcellular compartment, nuclear or cytoplasmic, may greatly differ. The usual methods of RNA isolation rely on harsh chemicals, e.g. phenol, guanidine thiocyanate and chloroform, to denature protein and create a lysate enriched in nuclei acids. The earliest protocols featured multiple steps, including removal of nuclei by pelleting from a cellular homogenate, and isolation of RNA from cytoplasm alone. Over the years, multi-step approaches were largely replaced by one-step ⁎ Tel.: +1 713 834 6268; fax: +1 713 834 6273. E-mail address: [email protected]. 0167-4781/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbaexp.2006.08.006
procedures, using mixtures of appropriate denaturants, to yield what is termed “total RNA” derived from combined nuclear and cytoplasmic RNA populations. The prevailing dogma, established in the 1970s, is that cytoplasmic RNA represents the vast majority of cellular RNA species, with nuclear-localized RNA contributing less than 1% of total RNA. Therefore, both approaches, fractionation of cytoplasm and without fractionation, to RNA isolation are used interchangeably to obtain a product loosely defined as “total RNA”. In the work by Schwanekamp et al., separation of nuclei and isolation of RNA from nuclear and cytoplasmic fractions of cells preceded use of these individual RNA populations as probes for microarray analysis [1]. The purpose of these analyses was to determine time-dependent effects of dioxin (TCDD) exposure of normal, nontransformed cells (mouse embryonic fibroblast cells or MEFs), by assaying levels and spatial distribution of RNA. TCDD acts as a ligand, binding to the aryl hydrocarbon receptor (AHR) to induce nuclear translocation, interaction between AHR and AHR nuclear translocator (ARNT) and binding of a TCCD–AHR–ARNT complex to sequence-specific, upstream regulatory elements, in a manner similar to nuclear receptor action at target genes [2,3]. Surprisingly, comparison of gene expression patterns by global, microarray approaches revealed highly discordant patterns of RNA expression in the nucleus and cytoplasm. According to these analyses, there was little support for a direct, substrate–
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M.C. Barton / Biochimica et Biophysica Acta 1759 (2006) 385–387
Fig. 1. Positive and negative influences on RNA levels. The pre-mRNA chain (gray) emerging from RNA Polymerase II is capped (red), which promotes continuation of elongation. Splicing initiates with association of splicing factors, RNA and the CTD. RNA is protected at the 3′ end by polyadenylation. Quality control mechanisms act prior to nuclear export, and exosome-mediated degradation of RNA may occur. Within the cytoplasm, bulk mRNA turnover occurs, which may include micro-RNA targeting and/or NMDA to degrade mRNA, in opposition to mechanisms that act to increase stability.
product relationship linking nuclear to cytoplasmic RNAs, encoded by the same gene. Comparisons of the ratio between nuclear RNA and cytoplasmic RNA levels show that highly expressed, nuclear-derived RNAs may represent 50%–70% of the sum of gene-specific nuclear and cytoplasmic RNA or “total RNA”. Differences between nuclear and cytoplasmic RNA levels of specific genes, due to regulated processing of pre-mRNA and cytoplasmic mRNA, are not unexpected but the degree of divergence between nuclear and cytoplasmic response in this study suggests that a “dioxin-regulated response” may encompass regulatory events not previously appreciated [4]. RNA turnover can take place in the cytoplasm, the nucleus or both compartments (Fig. 1). Multiple mechanisms affecting the final concentration, sequence and quality of pre-mRNA are invoked as nascent RNA transcripts emerge from the large subunit of RNA Polymerase II [5]. Capping of the 5′ end of RNA and splicing are coordinated with elongation of transcription by interactions between RNA, RNA Polymerase II CTD (carboxyterminal domain), elongation factors and specific processing proteins [6,7]. Quality control measures in both the nucleus and cytoplasm are taken to degrade RNA that doesn’t meet strict standards of surveillance pathways [8,9]. Before defining the impact of these mechanisms in a given regulatory response, specific information should be gathered. First, a determination of genes directly regulated by transcription factor complexes, e.g., TCCD–AHR complexes, should be undertaken by probing genomic sequences with DNA fragments precipitated by chromatin immunoprecipitation or ChIPon-chip analysis. This approach reveals primary gene targets, versus those affected by secondary responses to a given stimulus [10]. Selected, direct gene targets can be evaluated by nuclear run-on approaches to determine which are regulated by initiation of transcription, at least in the initial stages of gene expression. The impact of mechanisms controlling cytoplasmic
message stability of these gene products is estimated by measurements of RNA decay after transcription inhibition. Both run-on and decay measurements are adaptable to global, microarray analyses but a few well-chosen genes, analyzed individually, may offer considerable insight into potential control mechanisms. Changes in the design of the microarray platform and reevaluation of expression patterns may be especially valuable. For example, a majority of genes in the study under discussion are represented on the microarray by a single 70-nucleotide probe [1]. Hybridization of multiple targets, both exonic and intronic, along the length of specific genes may reveal unexpected changes or imbalances in transcript elongation, splicing or export. Transcription, splicing and processing are tightly coupled in the nucleus and optimal efficiency of each is co-dependent, offering potential “checkpoints” to ensure a wellintegrated process [6]. These checkpoints may detect aberrantly spliced or formed RNA species and promote degradation. Similarly, lack of 3′ polyadenylation or loss of a 5′ cap exposes pre-mRNA to exosome-mediated degradation [8,9]. Interestingly, among the most highly expressed genes, present in the nuclear TCDD-response group, are several encoding nucleicacid binding proteins that could contribute to RNA processing [1]. How their presence as enriched nuclear RNA species leads indirectly to the observed profiles of TCDD-response RNAs remains to be determined. In the theme of re-probing a differently designed microarray platform, analysis of micro-RNA presence may be conducted. Micro-RNAs regulate levels of RNA by RNAi-mediated methods of targeted destruction or by inhibition of translation [11]. The latter may promote alterations in ribosome association with mRNA, a process that also affects message stability. Along with RNA surveillance by Nonsense-Mediated Decay (NMD), these pathways can ultimately alter the ratio between nuclear and cytoplasmic RNA species [12]. Overall, the studies discussed here show that “total RNA” includes RNA species that are never translated to gene product and never realized as a functional output, especially specific genes over-represented among nuclear RNA species. While RNA turnover has been known for many years, the extent of post-transcription processes that alter nascent RNA may not be appreciated when a regulatory response is defined by global expression analysis. Multiple microarray platforms, careful isolation of specific fractions of compartmentalized RNA populations and determination of direct gene targets are important steps that can be used in global approaches to understanding regulated maturation of RNA. References [1] J.A. Schwanekamp, M.A. Sartor, S. Karyala, D. Halbleib, M. Medvedovic, C.R. Tomlinson, Biochim. Biophys. Acta 1759 (2006) 388–402. [2] E.C. Hoffman, H. Reyes, F.F. Chu, F. Sander, L.H. Conley, B.A. Brooks, O. Hankinson, Science 252 (1991) 954–958. [3] J.P. Whitlock Jr., Chem. Res. Toxicol. 6 (1993) 754–763. [4] M.C. Kohn, N.J. Walker, A.H. Kim, C.J. Portier, Toxicology 162 (2001) 193–208. [5] N.A. Woychik, M. Hampsey, Cell 108 (2002) 453–463.
M.C. Barton / Biochimica et Biophysica Acta 1759 (2006) 385–387 [6] D.L. Bentley, Curr. Opin. Cell Biol. 17 (2005) 251–256. [7] R.J. Sims III, R. Belotserkovskaya, D. Reinberg, Genes Dev. 18 (2004) 2437–2468. [8] C. Saguez, J.R. Olesen, T.H. Jensen, Curr. Opin. Cell Biol. 17 (2005) 287–293. [9] P. Sommer, U. Nehrbass, Curr. Opin. Cell Biol. 17 (2005) 294–301.
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[10] A.S. Weinmann, P.S. Yan, M.J. Oberley, T.H. Huang, P.J. Farnham, Genes Dev. 16 (2002) 235–244. [11] M.A. Valencia-Sanchez, J. Liu, G.J. Hannon, R. Parker, Genes Dev. 20 (2006) 515–524. [12] J.P. Gudikote, J.S. Imam, R.F. Garcia, M.F. Wilkinson, Nat. Struct. Mol. Biol. 12 (2005) 801–809.
Review
Influences along the path to maturity: Regulation of cellular levels of RNA Michelle Craig Barton ⁎ Department of Biochemistry and Molecular Biology, Program in Genes and Development, Graduate School of Biomedical Sciences, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA Received 28 August 2006; accepted 29 August 2006 Available online 5 September 2006
Abstract Initiation of RNA transcription may be a rate-limiting step in gene expression but it is only the first of many regulatory processes that impinge on nascent RNA along its path to maturity. Discontinuity between gene expression patterns within the nucleus and the cytoplasm suggests that multiple post-transcription regulatory points greatly influence the final RNA product, even to the extent of dramatically shifting the gene targets identified as a defined regulatory response. © 2006 Elsevier B.V. All rights reserved. Keywords: Microarray; Dioxin; RNA; Transcription; Processing
In gene expression, the number of regulatory controls between signal input and functional outcome comprises a long and, seemingly, ever-expanding list. To initiate the process of understanding the regulatory impact of each control point, the simple task of measuring steady-state levels of RNA, encoded by a given gene, is generally used as a first estimate of regulatory response. Recent results reported by Tomlinson and colleagues [1], in this issue of Biochimica et Biophysica Acta, bring into question whether even this task is so simple. Two major points are made in this article: (1) Global expression patterns are established by numerous processes that act on nascent RNA to alter its sequence, its localization and even its existence. (2) The functional term “total RNA” requires more precise definition, as the population of RNAs present in each subcellular compartment, nuclear or cytoplasmic, may greatly differ. The usual methods of RNA isolation rely on harsh chemicals, e.g. phenol, guanidine thiocyanate and chloroform, to denature protein and create a lysate enriched in nuclei acids. The earliest protocols featured multiple steps, including removal of nuclei by pelleting from a cellular homogenate, and isolation of RNA from cytoplasm alone. Over the years, multi-step approaches were largely replaced by one-step ⁎ Tel.: +1 713 834 6268; fax: +1 713 834 6273. E-mail address: [email protected]. 0167-4781/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbaexp.2006.08.006
procedures, using mixtures of appropriate denaturants, to yield what is termed “total RNA” derived from combined nuclear and cytoplasmic RNA populations. The prevailing dogma, established in the 1970s, is that cytoplasmic RNA represents the vast majority of cellular RNA species, with nuclear-localized RNA contributing less than 1% of total RNA. Therefore, both approaches, fractionation of cytoplasm and without fractionation, to RNA isolation are used interchangeably to obtain a product loosely defined as “total RNA”. In the work by Schwanekamp et al., separation of nuclei and isolation of RNA from nuclear and cytoplasmic fractions of cells preceded use of these individual RNA populations as probes for microarray analysis [1]. The purpose of these analyses was to determine time-dependent effects of dioxin (TCDD) exposure of normal, nontransformed cells (mouse embryonic fibroblast cells or MEFs), by assaying levels and spatial distribution of RNA. TCDD acts as a ligand, binding to the aryl hydrocarbon receptor (AHR) to induce nuclear translocation, interaction between AHR and AHR nuclear translocator (ARNT) and binding of a TCCD–AHR–ARNT complex to sequence-specific, upstream regulatory elements, in a manner similar to nuclear receptor action at target genes [2,3]. Surprisingly, comparison of gene expression patterns by global, microarray approaches revealed highly discordant patterns of RNA expression in the nucleus and cytoplasm. According to these analyses, there was little support for a direct, substrate–
386
M.C. Barton / Biochimica et Biophysica Acta 1759 (2006) 385–387
Fig. 1. Positive and negative influences on RNA levels. The pre-mRNA chain (gray) emerging from RNA Polymerase II is capped (red), which promotes continuation of elongation. Splicing initiates with association of splicing factors, RNA and the CTD. RNA is protected at the 3′ end by polyadenylation. Quality control mechanisms act prior to nuclear export, and exosome-mediated degradation of RNA may occur. Within the cytoplasm, bulk mRNA turnover occurs, which may include micro-RNA targeting and/or NMDA to degrade mRNA, in opposition to mechanisms that act to increase stability.
product relationship linking nuclear to cytoplasmic RNAs, encoded by the same gene. Comparisons of the ratio between nuclear RNA and cytoplasmic RNA levels show that highly expressed, nuclear-derived RNAs may represent 50%–70% of the sum of gene-specific nuclear and cytoplasmic RNA or “total RNA”. Differences between nuclear and cytoplasmic RNA levels of specific genes, due to regulated processing of pre-mRNA and cytoplasmic mRNA, are not unexpected but the degree of divergence between nuclear and cytoplasmic response in this study suggests that a “dioxin-regulated response” may encompass regulatory events not previously appreciated [4]. RNA turnover can take place in the cytoplasm, the nucleus or both compartments (Fig. 1). Multiple mechanisms affecting the final concentration, sequence and quality of pre-mRNA are invoked as nascent RNA transcripts emerge from the large subunit of RNA Polymerase II [5]. Capping of the 5′ end of RNA and splicing are coordinated with elongation of transcription by interactions between RNA, RNA Polymerase II CTD (carboxyterminal domain), elongation factors and specific processing proteins [6,7]. Quality control measures in both the nucleus and cytoplasm are taken to degrade RNA that doesn’t meet strict standards of surveillance pathways [8,9]. Before defining the impact of these mechanisms in a given regulatory response, specific information should be gathered. First, a determination of genes directly regulated by transcription factor complexes, e.g., TCCD–AHR complexes, should be undertaken by probing genomic sequences with DNA fragments precipitated by chromatin immunoprecipitation or ChIPon-chip analysis. This approach reveals primary gene targets, versus those affected by secondary responses to a given stimulus [10]. Selected, direct gene targets can be evaluated by nuclear run-on approaches to determine which are regulated by initiation of transcription, at least in the initial stages of gene expression. The impact of mechanisms controlling cytoplasmic
message stability of these gene products is estimated by measurements of RNA decay after transcription inhibition. Both run-on and decay measurements are adaptable to global, microarray analyses but a few well-chosen genes, analyzed individually, may offer considerable insight into potential control mechanisms. Changes in the design of the microarray platform and reevaluation of expression patterns may be especially valuable. For example, a majority of genes in the study under discussion are represented on the microarray by a single 70-nucleotide probe [1]. Hybridization of multiple targets, both exonic and intronic, along the length of specific genes may reveal unexpected changes or imbalances in transcript elongation, splicing or export. Transcription, splicing and processing are tightly coupled in the nucleus and optimal efficiency of each is co-dependent, offering potential “checkpoints” to ensure a wellintegrated process [6]. These checkpoints may detect aberrantly spliced or formed RNA species and promote degradation. Similarly, lack of 3′ polyadenylation or loss of a 5′ cap exposes pre-mRNA to exosome-mediated degradation [8,9]. Interestingly, among the most highly expressed genes, present in the nuclear TCDD-response group, are several encoding nucleicacid binding proteins that could contribute to RNA processing [1]. How their presence as enriched nuclear RNA species leads indirectly to the observed profiles of TCDD-response RNAs remains to be determined. In the theme of re-probing a differently designed microarray platform, analysis of micro-RNA presence may be conducted. Micro-RNAs regulate levels of RNA by RNAi-mediated methods of targeted destruction or by inhibition of translation [11]. The latter may promote alterations in ribosome association with mRNA, a process that also affects message stability. Along with RNA surveillance by Nonsense-Mediated Decay (NMD), these pathways can ultimately alter the ratio between nuclear and cytoplasmic RNA species [12]. Overall, the studies discussed here show that “total RNA” includes RNA species that are never translated to gene product and never realized as a functional output, especially specific genes over-represented among nuclear RNA species. While RNA turnover has been known for many years, the extent of post-transcription processes that alter nascent RNA may not be appreciated when a regulatory response is defined by global expression analysis. Multiple microarray platforms, careful isolation of specific fractions of compartmentalized RNA populations and determination of direct gene targets are important steps that can be used in global approaches to understanding regulated maturation of RNA. References [1] J.A. Schwanekamp, M.A. Sartor, S. Karyala, D. Halbleib, M. Medvedovic, C.R. Tomlinson, Biochim. Biophys. Acta 1759 (2006) 388–402. [2] E.C. Hoffman, H. Reyes, F.F. Chu, F. Sander, L.H. Conley, B.A. Brooks, O. Hankinson, Science 252 (1991) 954–958. [3] J.P. Whitlock Jr., Chem. Res. Toxicol. 6 (1993) 754–763. [4] M.C. Kohn, N.J. Walker, A.H. Kim, C.J. Portier, Toxicology 162 (2001) 193–208. [5] N.A. Woychik, M. Hampsey, Cell 108 (2002) 453–463.
M.C. Barton / Biochimica et Biophysica Acta 1759 (2006) 385–387 [6] D.L. Bentley, Curr. Opin. Cell Biol. 17 (2005) 251–256. [7] R.J. Sims III, R. Belotserkovskaya, D. Reinberg, Genes Dev. 18 (2004) 2437–2468. [8] C. Saguez, J.R. Olesen, T.H. Jensen, Curr. Opin. Cell Biol. 17 (2005) 287–293. [9] P. Sommer, U. Nehrbass, Curr. Opin. Cell Biol. 17 (2005) 294–301.
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[10] A.S. Weinmann, P.S. Yan, M.J. Oberley, T.H. Huang, P.J. Farnham, Genes Dev. 16 (2002) 235–244. [11] M.A. Valencia-Sanchez, J. Liu, G.J. Hannon, R. Parker, Genes Dev. 20 (2006) 515–524. [12] J.P. Gudikote, J.S. Imam, R.F. Garcia, M.F. Wilkinson, Nat. Struct. Mol. Biol. 12 (2005) 801–809.