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Inositol pyrophosphates regulate RNA polymerase I-mediated rRNA transcription in Saccharomyces cerevisiae.

Thota SG, Unnikannan CP, Thampatty SR, Manorama R, Bhandari R - Biochem. J. (2015)

Bottom Line: We examined whether inositol pyrophosphates, energy-rich derivatives of inositol that act as metabolic messengers, play a role in ribosome synthesis in the budding yeast, Saccharomyces cerevisiae.We determined that the Pol I subunits, A190, A43 and A34.5, can accept a β-phosphate moiety from inositol pyrophosphates to undergo serine pyrophosphorylation.Taken together, our findings highlight inositol pyrophosphates as novel regulators of rRNA transcription.

View Article: PubMed Central - PubMed

Affiliation: *Laboratory of Cell Signalling, Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad, Telangana, India.

ABSTRACT
Ribosome biogenesis is an essential cellular process regulated by the metabolic state of a cell. We examined whether inositol pyrophosphates, energy-rich derivatives of inositol that act as metabolic messengers, play a role in ribosome synthesis in the budding yeast, Saccharomyces cerevisiae. Yeast strains lacking the inositol hexakisphosphate (IP6) kinase Kcs1, which is required for the synthesis of inositol pyrophosphates, display increased sensitivity to translation inhibitors and decreased protein synthesis. These phenotypes are reversed on expression of enzymatically active Kcs1, but not on expression of the inactive form. The kcs1Δ yeast cells exhibit reduced levels of ribosome subunits, suggesting that they are defective in ribosome biogenesis. The rate of rRNA synthesis, the first step of ribosome biogenesis, is decreased in kcs1Δ yeast strains, suggesting that RNA polymerase I (Pol I) activity may be reduced in these cells. We determined that the Pol I subunits, A190, A43 and A34.5, can accept a β-phosphate moiety from inositol pyrophosphates to undergo serine pyrophosphorylation. Although there is impaired rRNA synthesis in kcs1Δ yeast cells, we did not find any defect in recruitment of Pol I on rDNA, but observed that the rate of transcription elongation was compromised. Taken together, our findings highlight inositol pyrophosphates as novel regulators of rRNA transcription.

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IP7 pyrophosphorylates RNA Pol I subunits(A) Purified, GST-tagged, full-length (FL) proteins Uaf30, A34.5, and A43, and the indicated fragments of A135 and A190, were incubated with 5[β-32P]IP7. Proteins were resolved using NuPAGE and transferred to a PVDF membrane. Pyrophosphorylation was detected by phosphorimager scanning (right) and proteins were detected by Western blotting (left). (B) Purified, GST-tagged, A190 fragments were pyrophosphorylated as in (A). (C) Purified GST-tagged A190 fragments corresponding to the native sequence and the indicated serine-to-alanine point mutants were pyrophosphorylated as in (A). (D–F) Pyrophosphorylation, as in (A), of purified, GST-tagged, FL fragments, and the indicated serine-to-alanine point mutants of A43. (G) Pyrophosphorylation, as in (A), of purified GST-tagged FL in-frame deletion, a C-terminally truncated fragment and the indicated serine-to-alanine point mutants of A34.5. To improve visualization, phosphorimager scans in (A), (F) and (G) were subjected to tonal range adjustment of the whole image using Adobe Photoshop (level adjustment). The start and end amino acid numbers of protein fragments are indicated in brackets. The dividing lines between lanes in panels (A) and (F) indicate the removal of non-essential lanes from a single original gel.
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Figure 4: IP7 pyrophosphorylates RNA Pol I subunits(A) Purified, GST-tagged, full-length (FL) proteins Uaf30, A34.5, and A43, and the indicated fragments of A135 and A190, were incubated with 5[β-32P]IP7. Proteins were resolved using NuPAGE and transferred to a PVDF membrane. Pyrophosphorylation was detected by phosphorimager scanning (right) and proteins were detected by Western blotting (left). (B) Purified, GST-tagged, A190 fragments were pyrophosphorylated as in (A). (C) Purified GST-tagged A190 fragments corresponding to the native sequence and the indicated serine-to-alanine point mutants were pyrophosphorylated as in (A). (D–F) Pyrophosphorylation, as in (A), of purified, GST-tagged, FL fragments, and the indicated serine-to-alanine point mutants of A43. (G) Pyrophosphorylation, as in (A), of purified GST-tagged FL in-frame deletion, a C-terminally truncated fragment and the indicated serine-to-alanine point mutants of A34.5. To improve visualization, phosphorimager scans in (A), (F) and (G) were subjected to tonal range adjustment of the whole image using Adobe Photoshop (level adjustment). The start and end amino acid numbers of protein fragments are indicated in brackets. The dividing lines between lanes in panels (A) and (F) indicate the removal of non-essential lanes from a single original gel.

Mentions: Sensitivity to translation inhibitors was observed in kcs1Δ strain, which displays no detectable inositol pyrophosphates, but not in vip1Δ or kcs1Δddp1Δ strains, which have only 5-IP7 or 1-IP7, respectively (see Figure 1A). Therefore, protein pyrophosphorylation, which can be effected by either form of IP7, may be the underlying molecular basis for the regulation of rDNA transcription by inositol pyrophosphates, and the target may be RNA Pol I and associated factors involved in rRNA synthesis. Regulation of Pol I activity during the logarithmic phase of growth occurs at the level of promoter binding [2] or transcription elongation [39]. Recruitment of RNA Pol I to the rDNA promoter involves four transcription factors, including the upstream activating factor (UAF) complex, TATA-binding protein (TBP), core factor (CF) complex and Rrn3 [40]. Transcription elongation is brought about by the 590-kDa 14-subunit RNA Pol I complex [1]. We examined databases curating mapped phosphorylation sites on these proteins, including PhosphoGRID (http://www.phosphogrid.org) and PhosphoPep (http://www.phosphopep.org), and picked those proteins that contain a pyrophosphorylation consensus site, i.e. two or more serine residues surrounded by acidic amino acids [17,22]. We narrowed the list down to five proteins containing an acidic serine motif: Uaf30, which is part of the UAF complex; and A190, A135, A43 and A34.5, which belong to the RNA Pol I elongation complex (see Supplementary Figure S2). We expressed these proteins in yeast as fusions to GST and examined their pyrophosphorylation with radiolabelled IP7. As A190 and A135 are large proteins of 186 kDa and 135 kDa, respectively, we tested only fragments of these proteins containing the predicted pyrophosphorylation sites. A43 and A34.5 are pyrophosphorylated by IP7, as is the C-terminal A190 fragment (Figure 4A). Uaf30 and an N-terminal fragment of A135, both of which contain potential pyrophosphorylation sites (see Supplementary Figures S2A and S2B), were found not to be IP7 substrates.


Inositol pyrophosphates regulate RNA polymerase I-mediated rRNA transcription in Saccharomyces cerevisiae.

Thota SG, Unnikannan CP, Thampatty SR, Manorama R, Bhandari R - Biochem. J. (2015)

IP7 pyrophosphorylates RNA Pol I subunits(A) Purified, GST-tagged, full-length (FL) proteins Uaf30, A34.5, and A43, and the indicated fragments of A135 and A190, were incubated with 5[β-32P]IP7. Proteins were resolved using NuPAGE and transferred to a PVDF membrane. Pyrophosphorylation was detected by phosphorimager scanning (right) and proteins were detected by Western blotting (left). (B) Purified, GST-tagged, A190 fragments were pyrophosphorylated as in (A). (C) Purified GST-tagged A190 fragments corresponding to the native sequence and the indicated serine-to-alanine point mutants were pyrophosphorylated as in (A). (D–F) Pyrophosphorylation, as in (A), of purified, GST-tagged, FL fragments, and the indicated serine-to-alanine point mutants of A43. (G) Pyrophosphorylation, as in (A), of purified GST-tagged FL in-frame deletion, a C-terminally truncated fragment and the indicated serine-to-alanine point mutants of A34.5. To improve visualization, phosphorimager scans in (A), (F) and (G) were subjected to tonal range adjustment of the whole image using Adobe Photoshop (level adjustment). The start and end amino acid numbers of protein fragments are indicated in brackets. The dividing lines between lanes in panels (A) and (F) indicate the removal of non-essential lanes from a single original gel.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4325516&req=5

Figure 4: IP7 pyrophosphorylates RNA Pol I subunits(A) Purified, GST-tagged, full-length (FL) proteins Uaf30, A34.5, and A43, and the indicated fragments of A135 and A190, were incubated with 5[β-32P]IP7. Proteins were resolved using NuPAGE and transferred to a PVDF membrane. Pyrophosphorylation was detected by phosphorimager scanning (right) and proteins were detected by Western blotting (left). (B) Purified, GST-tagged, A190 fragments were pyrophosphorylated as in (A). (C) Purified GST-tagged A190 fragments corresponding to the native sequence and the indicated serine-to-alanine point mutants were pyrophosphorylated as in (A). (D–F) Pyrophosphorylation, as in (A), of purified, GST-tagged, FL fragments, and the indicated serine-to-alanine point mutants of A43. (G) Pyrophosphorylation, as in (A), of purified GST-tagged FL in-frame deletion, a C-terminally truncated fragment and the indicated serine-to-alanine point mutants of A34.5. To improve visualization, phosphorimager scans in (A), (F) and (G) were subjected to tonal range adjustment of the whole image using Adobe Photoshop (level adjustment). The start and end amino acid numbers of protein fragments are indicated in brackets. The dividing lines between lanes in panels (A) and (F) indicate the removal of non-essential lanes from a single original gel.
Mentions: Sensitivity to translation inhibitors was observed in kcs1Δ strain, which displays no detectable inositol pyrophosphates, but not in vip1Δ or kcs1Δddp1Δ strains, which have only 5-IP7 or 1-IP7, respectively (see Figure 1A). Therefore, protein pyrophosphorylation, which can be effected by either form of IP7, may be the underlying molecular basis for the regulation of rDNA transcription by inositol pyrophosphates, and the target may be RNA Pol I and associated factors involved in rRNA synthesis. Regulation of Pol I activity during the logarithmic phase of growth occurs at the level of promoter binding [2] or transcription elongation [39]. Recruitment of RNA Pol I to the rDNA promoter involves four transcription factors, including the upstream activating factor (UAF) complex, TATA-binding protein (TBP), core factor (CF) complex and Rrn3 [40]. Transcription elongation is brought about by the 590-kDa 14-subunit RNA Pol I complex [1]. We examined databases curating mapped phosphorylation sites on these proteins, including PhosphoGRID (http://www.phosphogrid.org) and PhosphoPep (http://www.phosphopep.org), and picked those proteins that contain a pyrophosphorylation consensus site, i.e. two or more serine residues surrounded by acidic amino acids [17,22]. We narrowed the list down to five proteins containing an acidic serine motif: Uaf30, which is part of the UAF complex; and A190, A135, A43 and A34.5, which belong to the RNA Pol I elongation complex (see Supplementary Figure S2). We expressed these proteins in yeast as fusions to GST and examined their pyrophosphorylation with radiolabelled IP7. As A190 and A135 are large proteins of 186 kDa and 135 kDa, respectively, we tested only fragments of these proteins containing the predicted pyrophosphorylation sites. A43 and A34.5 are pyrophosphorylated by IP7, as is the C-terminal A190 fragment (Figure 4A). Uaf30 and an N-terminal fragment of A135, both of which contain potential pyrophosphorylation sites (see Supplementary Figures S2A and S2B), were found not to be IP7 substrates.

Bottom Line: We examined whether inositol pyrophosphates, energy-rich derivatives of inositol that act as metabolic messengers, play a role in ribosome synthesis in the budding yeast, Saccharomyces cerevisiae.We determined that the Pol I subunits, A190, A43 and A34.5, can accept a β-phosphate moiety from inositol pyrophosphates to undergo serine pyrophosphorylation.Taken together, our findings highlight inositol pyrophosphates as novel regulators of rRNA transcription.

View Article: PubMed Central - PubMed

Affiliation: *Laboratory of Cell Signalling, Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad, Telangana, India.

ABSTRACT
Ribosome biogenesis is an essential cellular process regulated by the metabolic state of a cell. We examined whether inositol pyrophosphates, energy-rich derivatives of inositol that act as metabolic messengers, play a role in ribosome synthesis in the budding yeast, Saccharomyces cerevisiae. Yeast strains lacking the inositol hexakisphosphate (IP6) kinase Kcs1, which is required for the synthesis of inositol pyrophosphates, display increased sensitivity to translation inhibitors and decreased protein synthesis. These phenotypes are reversed on expression of enzymatically active Kcs1, but not on expression of the inactive form. The kcs1Δ yeast cells exhibit reduced levels of ribosome subunits, suggesting that they are defective in ribosome biogenesis. The rate of rRNA synthesis, the first step of ribosome biogenesis, is decreased in kcs1Δ yeast strains, suggesting that RNA polymerase I (Pol I) activity may be reduced in these cells. We determined that the Pol I subunits, A190, A43 and A34.5, can accept a β-phosphate moiety from inositol pyrophosphates to undergo serine pyrophosphorylation. Although there is impaired rRNA synthesis in kcs1Δ yeast cells, we did not find any defect in recruitment of Pol I on rDNA, but observed that the rate of transcription elongation was compromised. Taken together, our findings highlight inositol pyrophosphates as novel regulators of rRNA transcription.

Show MeSH
Related in: MedlinePlus