RESUMO
Fungal Trl1 is an essential trifunctional tRNA splicing enzyme that heals and seals tRNA exons with 2',3'-cyclic-PO4 and 5'-OH ends. Trl1 is composed of C-terminal cyclic phosphodiesterase and central polynucleotide kinase end-healing domains that generate the 3'-OH,2'-PO4 and 5'-PO4 termini required for sealing by an N-terminal ATP-dependent ligase domain. Trl1 enzymes are present in many human fungal pathogens and are promising targets for antifungal drug discovery because their domain structures and biochemical mechanisms are unique compared to the mammalian RtcB-type tRNA splicing enzyme. Here we report that Mucorales species (deemed high-priority human pathogens by WHO) elaborate a noncanonical tRNA splicing apparatus in which a monofunctional RNA ligase enzyme is encoded separately from any end-healing enzymes. We show that Mucor circinelloides RNA ligase (MciRNL) is active in tRNA splicing in vivo in budding yeast in lieu of the Trl1 ligase domain. Biochemical and kinetic characterization of recombinant MciRNL underscores its requirement for a 2'-PO4 terminus in the end-joining reaction, whereby the 2'-PO4 enhances the rates of RNA 5'-adenylylation (step 2) and phosphodiester synthesis (step 3) by â¼125-fold and â¼6200-fold, respectively. In the canonical fungal tRNA splicing pathway, the splice junction 2'-PO4 installed by RNA ligase is removed by a dedicated NAD+-dependent RNA 2'-phosphotransferase Tpt1. Here we identify and affirm by genetic complementation in yeast the biological activity of Tpt1 orthologs from three Mucorales species. Recombinant M. circinelloides Tpt1 has vigorous NAD+-dependent RNA 2'-phosphotransferase activity in vitro.
Assuntos
Mucorales , Animais , Humanos , Mucorales/genética , Mucorales/metabolismo , NAD/metabolismo , RNA/genética , RNA de Transferência/genética , RNA de Transferência/metabolismo , RNA Ligase (ATP)/genética , RNA Ligase (ATP)/metabolismo , Saccharomyces cerevisiae/metabolismo , Ligases , Polinucleotídeo 5'-Hidroxiquinase/química , Splicing de RNA , Mamíferos/genéticaRESUMO
Starvation of Schizosaccharomyces pombe for inorganic phosphate elicits adaptive transcriptome changes in which mRNAs driving ribosome biogenesis, tRNA biogenesis, and translation are globally downregulated, while those for autophagy and phosphate mobilization are upregulated. Here, we interrogated three components of the starvation response: upregulated autophagy; the role of transcription factor Pho7 (an activator of the PHO regulon); and upregulated expression of ecl3, one of three paralogous genes (ecl1, ecl2, and ecl3) collectively implicated in cell survival during other nutrient stresses. Ablation of autophagy factor Atg1 resulted in early demise of phosphate-starved fission yeast, as did ablation of Pho7. Transcriptome profiling of phosphate-starved pho7Δ cells highlighted Pho7 as an activator of genes involved in phosphate acquisition and mobilization, not limited to the original three-gene PHO regulon, and additional starvation-induced genes (including ecl3) not connected to phosphate dynamics. Pho7-dependent gene induction during phosphate starvation tracked with the presence of Pho7 DNA-binding elements in the gene promoter regions. Fewer ribosome protein genes were downregulated in phosphate-starved pho7Δ cells versus WT, which might contribute to their shortened lifespan. An ecl3Δ mutant elicited no gene expression changes in phosphate-replete cells and had no impact on survival during phosphate starvation. By contrast, pan-ecl deletion (ecl123Δ) curtailed lifespan during chronic phosphate starvation. Phosphate-starved ecl123Δ cells experienced a more widespread downregulation of mRNAs encoding aminoacyl tRNA synthetases vis-à-vis WT or pho7Δ cells. Collectively, these results enhance our understanding of fission yeast phosphate homeostasis and survival during nutrient deprivation.
Assuntos
Proteínas de Ligação a DNA , Longevidade , Fosfatos , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Fatores de Transcrição , Regulação Fúngica da Expressão Gênica , Longevidade/genética , Fosfatos/deficiência , RNA de Transferência/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Transcriptoma , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Expression of fission yeast Pho1 acid phosphatase is repressed under phosphate-replete conditions by transcription of an upstream prt lncRNA that interferes with the pho1 mRNA promoter. lncRNA-mediated interference is alleviated by genetic perturbations that elicit precocious lncRNA 3'-processing and transcription termination, such as (i) the inositol pyrophosphate pyrophosphatase-defective asp1-H397A allele, which results in elevated levels of IP8, and (ii) absence of the 14-3-3 protein Rad24. Combining rad24Δ with asp1-H397A causes a severe synthetic growth defect. A forward genetic screen for SRA (Suppressor of Rad24 Asp1-H397A) mutations identified a novel missense mutation (Tyr86Asp) of Pla1, the essential poly(A) polymerase subunit of the fission yeast cleavage and polyadenylation factor (CPF) complex. The pla1-Y86D allele was viable but slow-growing in an otherwise wild-type background. Tyr86 is a conserved active site constituent that contacts the RNA primer 3' nt and the incoming ATP. The Y86D mutation elicits a severe catalytic defect in RNA-primed poly(A) synthesis in vitro and in binding to an RNA primer. Yet, analyses of specific mRNAs indicate that poly(A) tails in pla1-Y86D cells are not different in size than those in wild-type cells, suggesting that other RNA interactors within CPF compensate for the defects of isolated Pla1-Y86D. Transcriptome profiling of pla1-Y86D cells revealed the accumulation of multiple RNAs that are normally rapidly degraded by the nuclear exosome under the direction of the MTREC complex, with which Pla1 associates. We suggest that Pla1-Y86D is deficient in the hyperadenylation of MTREC targets that precedes their decay by the exosome.
Assuntos
RNA Longo não Codificante , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Domínio Catalítico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Longo não Codificante/genética , Mutação , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
Fission yeast phosphate homeostasis gene pho1 is actively repressed during growth in phosphate-rich medium by transcription in cis of a long noncoding (lnc) RNA from the 5' flanking prt(nc-pho1) gene. Pho1 expression is: (i) derepressed by genetic maneuvers that favor precocious lncRNA 3'-processing and termination, in response to DSR and PAS signals in prt; and (ii) hyperrepressed in genetic backgrounds that dampen 3'-processing/termination efficiency. Governors of 3'-processing/termination include the RNA polymerase CTD code, the CPF (cleavage and polyadenylation factor) complex, termination factors Seb1 and Rhn1, and the inositol pyrophosphate signaling molecule 1,5-IP8 Here, we present genetic and biochemical evidence that fission yeast Duf89, a metal-dependent phosphatase/pyrophosphatase, is an antagonist of precocious 3'-processing/termination. We show that derepression of pho1 in duf89Δ cells correlates with squelching the production of full-length prt lncRNA and is erased or attenuated by: (i) DSR/PAS mutations in prt; (ii) loss-of-function mutations in components of the 3'-processing and termination machinery; (iii) elimination of the CTD Thr4-PO4 mark; (iv) interdicting CTD prolyl isomerization by Pin1; (v) inactivating the Asp1 kinase that synthesizes IP8; and (vi) loss of the putative IP8 sensor Spx1. The findings that duf89Δ is synthetically lethal with pho1-derepressive mutations CTD-S7A and aps1Δ-and that this lethality is rescued by CTD-T4A, CPF/Rhn1/Pin1 mutations, and spx1Δ-implicate Duf89 more broadly as a collaborator in cotranscriptional regulation of essential fission yeast genes. The duf89-D252A mutation, which abolishes Duf89 phosphohydrolase activity, phenocopied duf89 +, signifying that duf89Δ phenotypes are a consequence of Duf89 protein absence, not absence of Duf89 catalysis.
Assuntos
RNA Longo não Codificante , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Transcrição Gênica , Homeostase/genética , Fosfatos/metabolismo , RNA Polimerase II/genética , Terminação da Transcrição GenéticaRESUMO
Inorganic phosphate is an essential nutrient acquired by cells from their environment. Here, we characterize the adaptative responses of fission yeast to chronic phosphate starvation, during which cells enter a state of quiescence, initially fully reversible upon replenishing phosphate after 2 days but resulting in gradual loss of viability during 4 weeks of starvation. Time-resolved analyses of changes in mRNA levels revealed a coherent transcriptional program in which phosphate dynamics and autophagy were upregulated, while the machineries for rRNA synthesis and ribosome assembly, and for tRNA synthesis and maturation, were downregulated in tandem with global repression of genes encoding ribosomal proteins and translation factors. Consistent with the transcriptome changes, proteome analysis highlighted global depletion of 102 ribosomal proteins. Concomitant with this ribosomal protein deficit, 28S and 18S rRNAs became vulnerable to site-specific cleavages that generated temporally stable rRNA fragments. The finding that Maf1, a repressor of RNA polymerase III transcription, was upregulated during phosphate starvation prompted a hypothesis that its activity might prolong lifespan of the quiescent cells by limiting production of tRNAs. Indeed, we found that deletion of maf1 results in precocious death of phosphate-starved cells via a distinctive starvation-induced pathway associated with tRNA overproduction and dysfunctional tRNA biogenesis.
Assuntos
Fosfatos , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Fosfatos/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Ribossômicas/genética , RNA de Transferência/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Transcrição GênicaRESUMO
Expression of fission yeast Pho1 acid phosphatase is repressed under phosphate-replete conditions by transcription of an upstream prt lncRNA that interferes with the pho1 mRNA promoter. lncRNA control of pho1 mRNA synthesis is influenced by inositol pyrophosphate (IPP) kinase Asp1, deletion of which results in pho1 hyper-repression. A forward genetic screen for ADS (Asp1 Deletion Suppressor) mutations identified the 14-3-3 protein Rad24 as a governor of phosphate homeostasis. Production of full-length interfering prt lncRNA was squelched in rad24Δ cells, concomitant with increased production of pho1 mRNA and increased Pho1 activity, while shorter precociously terminated non-interfering prt transcripts persisted. Epistasis analysis showed that pho1 de-repression by rad24Δ depends on: (i) 3'-processing and transcription termination factors CPF, Pin1, and Rhn1; and (ii) Threonine-4 of the Pol2 CTD. Combining rad24Δ with the IPP pyrophosphatase-dead asp1-H397A allele caused a severe synthetic growth defect that was ameliorated by loss-of-function mutations in CPF, Pin1, and Rhn1, and by CTD phospho-site mutations T4A and Y1F. Rad24 function in repressing pho1 was effaced by mutation of its phosphate-binding pocket. Our findings instate a new role for a 14-3-3 protein as an antagonist of precocious RNA 3'-processing/termination.
Assuntos
Proteínas 14-3-3/metabolismo , Proteínas de Ciclo Celular/metabolismo , Regulação Fúngica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Transcrição Gênica , Fosfatase Ácida/genética , Fosfatase Ácida/metabolismo , Sequência de Aminoácidos , Proteínas de Ciclo Celular/química , Mapeamento Cromossômico , Perfilação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/química , Modelos Moleculares , Mutagênese , Mutação , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Interferência de RNA , RNA Longo não Codificante/genética , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Deleção de Sequência , Relação Estrutura-Atividade , Mutações Sintéticas Letais , Terminação da Transcrição Genética , Sequenciamento Completo do GenomaRESUMO
The system of long noncoding RNA (lncRNA)-mediated transcriptional interference that represses fission yeast phosphate homoeostasis gene pho1 provides a sensitive readout of genetic influences on cotranscriptional 3'-processing and termination and a tool for discovery of regulators of this phase of the Pol2 transcription cycle. Here, we conducted a genetic screen for relief of transcriptional interference that unveiled a mechanism by which Pol2 termination is enhanced via a gain-of-function mutation, G476S, in the RNA-binding domain of an essential termination factor, Seb1. The genetic and physical evidence for gain-of-function is compelling: 1) seb1-G476S de-represses pho1 and tgp1, both of which are subject to lncRNA-mediated transcriptional interference; 2) seb1-G476S elicits precocious lncRNA transcription termination in response to lncRNA 5'-proximal poly(A) signals; 3) seb1-G476S derepression of pho1 is effaced by loss-of-function mutations in cleavage and polyadenylation factor (CPF) subunits and termination factor Rhn1; 4) synthetic lethality of seb1-G476S with pho1 derepressive mutants rpb1-CTD-S7A and aps1∆ is rescued by CPF/Rhn1 loss-of-function alleles; and 5) seb1-G476S elicits an upstream shift in poly(A) site preference in several messenger RNA genes. A crystal structure of the Seb1-G476S RNA-binding domain indicates potential for gain of contacts from Ser476 to RNA nucleobases. To our knowledge, this is a unique instance of a gain-of-function phenotype in a eukaryal transcription termination protein.
Assuntos
Proteínas Fúngicas/metabolismo , Mutação com Ganho de Função , Regulação Fúngica da Expressão Gênica/fisiologia , Schizosaccharomyces/metabolismo , Sobrevivência Celular , Difosfatos/metabolismo , Proteínas Fúngicas/genética , Subunidades Proteicas , RNA Longo não Codificante , Schizosaccharomyces/genética , Terminação da Transcrição GenéticaRESUMO
Fission yeast Cleavage and Polyadenylation Factor (CPF), a 13-subunit complex, executes the cotranscriptional 3' processing of RNA polymerase II (Pol2) transcripts that precedes transcription termination. The three-subunit DPS sub-complex of CPF, consisting of a PP1-type phosphoprotein phosphatase Dis2, a WD-repeat protein Swd22, and a putative phosphatase regulatory factor Ppn1, associates with the CPF core to form the holo-CPF assembly. Here we probed the functional, physical, and genetic interactions of DPS by focusing on the Ppn1 subunit, which mediates association of DPS with the core. Transcriptional profiling by RNA-seq defined limited but highly concordant sets of protein-coding genes that were dysregulated in ppn1Δ, swd22Δ and dis2Δ cells, which included the DPSΔ down-regulated phosphate homeostasis genes pho1 and pho84 that are controlled by lncRNA-mediated transcriptional interference. Essential and inessential modules of the 710-aa Ppn1 protein were defined by testing the effects of Ppn1 truncations in multiple genetic backgrounds in which Ppn1 is required for growth. An N-terminal 172-aa disordered region was dispensable and its deletion alleviated hypomorphic phenotypes caused by deleting C-terminal aa 640-710. A TFIIS-like domain (aa 173-330) was not required for viability but was important for Ppn1 activity in phosphate homeostasis. Distinct sites within Ppn1 for binding to Dis2 (spanning Ppn1 aa 506 to 532) and Swd22 (from Ppn1 aa 533 to 578) were demarcated by yeast two-hybrid assays. Dis2 interaction-defective missense mutants of full-length Ppn1 (that retained Swd22 interaction) were employed to show that binding to Dis2 (or its paralog Sds21) was necessary for Ppn1 biological activity. Ppn1 function was severely compromised by missense mutations that selectively affected its binding to Swd22.
Assuntos
Hidrolases Anidrido Ácido/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteína Fosfatase 1/metabolismo , Schizosaccharomyces/fisiologia , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Alelos , Sequência de Aminoácidos , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Modelos Biológicos , Mutação , Fenótipo , Fosforilação , Transcrição Gênica , Transcriptoma , Fatores de Poliadenilação e Clivagem de mRNA/químicaRESUMO
The carboxyl-terminal domain (CTD) of RNA polymerase II (Pol2) consists of tandem repeats of a consensus heptapeptide Y1 S2 P3 T4 S5 P6 S7 The CTD recruits numerous proteins that drive or regulate gene expression. The trafficking of CTD-interacting proteins is orchestrated by remodeling CTD primary structure via Ser/Thr/Tyr phosphorylation and proline cis-trans isomerization, which collectively inscribe a CTD code. The fission yeast CTD consists of 29 heptad repeats. To decipher the output of the fission yeast CTD code, we genetically manipulated CTD length and amino acid content and then gauged the effects of these changes on gene expression. Whereas deleting 11 consensus heptads has no obvious effect on fission yeast growth, RNA-seq revealed that 25% of the protein-coding transcripts were dysregulated by CTD truncation. We profiled the transcriptomes of full-length CTD mutants, in which: all Tyr1 residues were replaced by Phe; all Ser2, Thr4, or Ser7 positions were changed to Ala; and half of the essential CTD code "letters" Pro3, Ser5, and Pro6 were mutated to Ala. Overlapping RNA-seq profiles suggested that a quarter of the complement of up-regulated mRNAs and half of the down-regulated mRNAs seen in full-length CTD mutants might be attributable to a decrement in wild-type CTD heptad number. Concordant mutant-specific transcriptional profiles were observed for Y1F, S2A, and T4A cells, and for P6â¢P6A and S5â¢S5A cells, suggesting that Tyr1-Ser2-Thr4 and Ser5-Pro6 comprise distinct "words" in the fission yeast CTD code. The phosphate regulon, which is repressed by lncRNA-mediated transcription interference, is de-repressed by CTD mutations P6â¢P6A and S5â¢S5A. De-repression of pho1 in P6â¢P6A and S5â¢S5A cells depends on cleavage and polyadenylation factor subunits Swd22 and Ppn1 and transcription termination factor Rhn1, signifying that Pro6 and Ser5 mutations elicit precocious lncRNA 3'-processing/termination.
RESUMO
The enzyme Tpt1 is an essential agent of fungal tRNA splicing that removes an internal RNA 2'-PO4 generated by fungal tRNA ligase. Tpt1 performs a two-step reaction in which: (i) the 2'-PO4 attacks NAD+ to form an RNA-2'-phospho-(ADP-ribose) intermediate; and (ii) transesterification of the ADP-ribose O2'' to the RNA 2'-phosphodiester yields 2'-OH RNA and ADP-ribose-1'',2''-cyclic phosphate. Because Tpt1 does not participate in metazoan tRNA splicing, and Tpt1 knockout has no apparent impact on mammalian physiology, Tpt1 is considered a potential anti-fungal drug target. Here we characterize Tpt1 enzymes from four human fungal pathogens: Coccidioides immitis, the agent of Valley Fever; Aspergillus fumigatus and Candida albicans, which cause invasive, often fatal, infections in immunocompromised hosts; and Candida auris, an emerging pathogen that is resistant to current therapies. All four pathogen Tpt1s were active in vivo in complementing a lethal Saccharomyces cerevisiae tpt1∆ mutation and in vitro in NAD+-dependent conversion of a 2'-PO4 to a 2'-OH. The fungal Tpt1s utilized nicotinamide hypoxanthine dinucleotide as a substrate in lieu of NAD+, albeit with much lower affinity, whereas nicotinic acid adenine dinucleotide was ineffective. Fungal Tpt1s efficiently removed an internal ribonucleotide 2'-phosphate from an otherwise all-DNA substrate. Replacement of an RNA ribose-2'-PO4 nucleotide with arabinose-2'-PO4 diminished enzyme specific activity by ≥2000-fold and selectively slowed step 2 of the reaction pathway, resulting in transient accumulation of an ara-2'-phospho-ADP-ribosylated intermediate. Our results implicate the 2'-PO4 ribonucleotide as the principal determinant of fungal Tpt1 nucleic acid substrate specificity.
RESUMO
Fission yeast Erh1 exists in a complex with RNA-binding protein Mmi1. Deletion of erh1 up-regulates the phosphate homeostasis gene pho1, which is normally repressed by transcription in cis of a 5' flanking prt lncRNA. Here we present evidence that de-repression of pho1 by erh1Δ is achieved through precocious 3'-processing/termination of prt lncRNA synthesis, to wit: (i) erh1Δ does not affect the activity of the prt or pho1 promoters per se; (ii) de-repression by erh1Δ depends on CPF (cleavage and polyadenylation factor) subunits Ctf1, Dis2, Ssu72, Swd22, and Ppn1 and on termination factor Rhn1; (iii) de-repression requires synthesis by the Asp1 IPP kinase of inositol 1-pyrophosphates (1-IPPs); (iv) de-repression is effaced by mutating Thr4 of the RNA polymerase II CTD to alanine; and (v) erh1Δ exerts an additive effect on pho1 de-repression in combination with mutating CTD Ser7 to alanine and with deletion of the IPP pyrophosphatase Aps1. These findings point to Erh1 as an antagonist of lncRNA termination in the prt-pho1 axis. In contrast, in mmi1Δ cells there is a reduction in pho1 mRNA and increase in the formation of a prt-pho1 read-through transcript, consistent with Mmi1 being an agonist of prt termination. We envision that Erh1 acts as a brake on Mmi1's ability to promote CPF-dependent termination during prt lncRNA synthesis. Consistent with this idea, erh1Δ de-repression of pho1 was eliminated by mutating the Mmi1-binding sites in the prt lncRNA.
Assuntos
Fosfatase Ácida/genética , Proteínas de Transporte/genética , Regulação Fúngica da Expressão Gênica/genética , RNA Longo não Codificante/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Terminação da Transcrição Genética/fisiologia , Fosfatos de Inositol/genética , Regiões Promotoras Genéticas/genética , RNA Polimerase II/genética , RNA Mensageiro/genéticaRESUMO
Fission yeast phosphate homeostasis genes are repressed in phosphate-rich medium by transcription of upstream lncRNAs that interferes with activation of the flanking mRNA promoters. lncRNA control of PHO gene expression is influenced by the Thr4 phospho-site in the RNA polymerase II CTD and the 3' processing/termination factors CPF and Rhn1, mutations of which result in hyper-repression of the PHO regulon. Here, we performed a forward genetic screen for mutations that de-repress Pho1 acid phosphatase expression in CTD-T4A cells. Sequencing of 18 independent STF (Suppressor of Threonine Four) isolates revealed, in every case, a mutation in the C-terminal pyrophosphatase domain of Asp1, a bifunctional inositol pyrophosphate (IPP) kinase/pyrophosphatase that interconverts 5-IP7 and 1,5-IP8. Focused characterization of two STF strains identified 51 coding genes coordinately upregulated vis-à-vis the parental T4A strain, including all three PHO regulon genes (pho1, pho84, tgp1). Whereas these STF alleles-asp1-386(Stop) and asp1-493(Stop)-were lethal in a wild-type CTD background, they were viable in combination with mutations in CPF and Rhn1, in which context Pho1 was also de-repressed. Our findings implicate Asp1 pyrophosphatase in constraining 1,5-IP8 or 1-IP7 synthesis by Asp1 kinase, without which 1-IPPs can accumulate to toxic levels that elicit precocious termination by CPF/Rhn1.
Assuntos
Fosfatase Ácida/genética , Proteínas do Citoesqueleto/genética , DNA Polimerase II/genética , Fosfatos de Inositol/metabolismo , Mutação , RNA Longo não Codificante/genética , Proteínas de Schizosaccharomyces pombe/genética , Fosfatase Ácida/metabolismo , Domínio Catalítico , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , DNA Polimerase II/química , DNA Polimerase II/metabolismo , Regulação Fúngica da Expressão Gênica , Enzimas Multifuncionais , Pirofosfatases , Regulon , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Mutações Sintéticas Letais , Terminação da Transcrição Genética , Regulação para CimaRESUMO
The phosphorylation pattern of Pol2 CTD Y1S2P3T4S5P6S7 repeats comprises an informational code coordinating transcription and RNA processing. cis-trans isomerization of CTD prolines expands the scope of the code in ways that are not well understood. Here we address this issue via analysis of fission yeast peptidyl-prolyl isomerase Pin1. A pin1Δ allele that does not affect growth per se is lethal in the absence of cleavage-polyadenylation factor (CPF) subunits Ppn1 and Swd22 and elicits growth defects absent CPF subunits Ctf1 and Dis2 and termination factor Rhn1. Whereas CTD S2A, T4A, and S7A mutants thrive in combination with pin1Δ, a Y1F mutant does not, nor do CTD mutants in which half the Pro3 or Pro6 residues are replaced by alanine. Phosphate-acquisition genes pho1, pho84 and tgp1 are repressed by upstream lncRNAs and are sensitive to changes in lncRNA 3' processing/termination. pin1Δ hyper-represses PHO gene expression and erases the de-repressive effect of CTD-S7A. Transcriptional profiling delineated sets of 56 and 22 protein-coding genes that are down-regulated and up-regulated in pin1Δ cells, respectively, 77% and 100% of which are downregulated/upregulated when the cis-proline-dependent Ssu72 CTD phosphatase is inactivated. Our results implicate Pin1 as a positive effector of 3' processing/termination that acts via Ssu72.
Assuntos
Proteínas de Ciclo Celular/genética , Regulação Fúngica da Expressão Gênica , Peptidilprolil Isomerase de Interação com NIMA/genética , Peptidilprolil Isomerase/genética , Fosfoproteínas Fosfatases/genética , Processamento de Terminações 3' de RNA , Proteínas de Schizosaccharomyces pombe/genética , Terminação da Transcrição Genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Fator de Especificidade de Clivagem e Poliadenilação/genética , Deleção de Genes , Peptidilprolil Isomerase de Interação com NIMA/química , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Proteínas Nucleares/genética , Peptidilprolil Isomerase/química , Peptidilprolil Isomerase/metabolismo , Fosfatos/metabolismo , Fosforilação , Domínios Proteicos/genética , Pirofosfatases/genética , RNA Polimerase II/genética , RNA-Seq , Regulon , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Serina/metabolismo , Treonina/metabolismoRESUMO
Interactions between RNA guanylyltransferase (GTase) and the C-terminal domain (CTD) repeats of RNA polymerase II (Pol2) and elongation factor Spt5 are thought to orchestrate cotranscriptional capping of nascent mRNAs. The crystal structure of a fission yeast GTaseâ¢Pol2 CTD complex reveals a unique docking site on the nucleotidyl transferase domain for an 8-amino-acid Pol2 CTD segment, S5PPSYSPTS5P, bracketed by two Ser5-PO4 marks. Analysis of GTase mutations that disrupt the Pol2 CTD interface shows that at least one of the two Ser5-PO4-binding sites is required for cell viability and that each site is important for cell growth at 37°C. Fission yeast GTase binds the Spt5 CTD at a separate docking site in the OB-fold domain that captures the Trp4 residue of the Spt5 nonapeptide repeat T(1)PAW(4)NSGSK. A disruptive mutation in the Spt5 CTD-binding site of GTase is synthetically lethal with mutations in the Pol2 CTD-binding site, signifying that the Spt5 and Pol2 CTDs cooperate to recruit capping enzyme in vivo. CTD phosphorylation has opposite effects on the interaction of GTase with Pol2 (Ser5-PO4 is required for binding) versus Spt5 (Thr1-PO4 inhibits binding). We propose that the state of Thr1 phosphorylation comprises a binary "Spt5 CTD code" that is read by capping enzyme independent of and parallel to its response to the state of the Pol2 CTD.
Assuntos
Modelos Moleculares , Nucleotidiltransferases/metabolismo , RNA Polimerase II , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/enzimologia , Fatores de Elongação da Transcrição , Código Genético , Nucleotidiltransferases/química , Fosforilação , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , RNA Polimerase II/química , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Treonina/metabolismo , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismoRESUMO
Fungal tRNA ligase (Trl1) rectifies RNA breaks with 2',3'-cyclic-PO4 and 5'-OH termini. Trl1 consists of three catalytic modules: an N-terminal ligase (LIG) domain; a central polynucleotide kinase (KIN) domain; and a C-terminal cyclic phosphodiesterase (CPD) domain. Trl1 enzymes found in all human fungal pathogens are untapped targets for antifungal drug discovery. Here we report a 1.9 Å crystal structure of Trl1 KIN-CPD from the pathogenic fungus Candida albicans, which adopts an extended conformation in which separate KIN and CPD domains are connected by an unstructured linker. CPD belongs to the 2H phosphotransferase superfamily by dint of its conserved central concave ß sheet and interactions of its dual HxT motif histidines and threonines with phosphate in the active site. Additional active site motifs conserved among the fungal CPD clade of 2H enzymes are identified. We present structures of the Candida Trl1 KIN domain at 1.5 to 2.0 Å resolution-as apoenzyme and in complexes with GTPâ¢Mg2+, IDPâ¢PO4, and dGDPâ¢PO4-that highlight conformational switches in the G-loop (which recognizes the guanine base) and lid-loop (poised over the nucleotide phosphates) that accompany nucleotide binding.
Assuntos
Domínio Catalítico , Guanosina Trifosfato/metabolismo , RNA Ligase (ATP)/química , RNA Ligase (ATP)/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Candida albicans , Domínio Catalítico/genética , Cristalografia por Raios X , Modelos Moleculares , Nucleotidases/química , Polinucleotídeo 5'-Hidroxiquinase/química , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , RNA Ligase (ATP)/genética , Relação Estrutura-AtividadeRESUMO
Fission yeast phosphate acquisition genes pho1, pho84, and tgp1 are repressed in phosphate-rich medium by transcription of upstream lncRNAs. Here, we show that phosphate homeostasis is subject to metabolite control by inositol pyrophosphates (IPPs), exerted through the 3'-processing/termination machinery and the Pol2 CTD code. Increasing IP8 (via Asp1 IPP pyrophosphatase mutation) de-represses the PHO regulon and leads to precocious termination of prt lncRNA synthesis. pho1 de-repression by IP8 depends on cleavage-polyadenylation factor (CPF) subunits, termination factor Rhn1, and the Thr4 letter of the CTD code. pho1 de-repression by mutation of the Ser7 CTD letter depends on IP8. Simultaneous inactivation of the Asp1 and Aps1 IPP pyrophosphatases is lethal, but this lethality is suppressed by mutations of CPF subunits Ppn1, Swd22, Ssu72, and Ctf1 and CTD mutation T4A. Failure to synthesize IP8 (via Asp1 IPP kinase mutation) results in pho1 hyper-repression. Synthetic lethality of asp1Δ with Ppn1, Swd22, and Ssu72 mutations argues that IP8 plays an important role in essential 3'-processing/termination events, albeit in a manner genetically redundant to CPF. Transcriptional profiling delineates an IPP-responsive regulon composed of genes overexpressed when IP8 levels are increased. Our results establish a novel role for IPPs in cell physiology.
Assuntos
Fosfatase Ácida/genética , Regulação Fúngica da Expressão Gênica , Fosfatos de Inositol/metabolismo , Regulon , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Terminação da Transcrição Genética , Fosfatase Ácida/metabolismo , Fator de Especificidade de Clivagem e Poliadenilação/genética , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Proteínas do Citoesqueleto/deficiência , Proteínas do Citoesqueleto/genética , Deleção de Genes , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Enzimas Multifuncionais , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Pirofosfatases/deficiência , Pirofosfatases/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
The carboxy-terminal domain (CTD) code encrypted within the Y1S2P3T4S5P6S7 heptad repeats of RNA polymerase II (Pol2) is deeply rooted in eukaryal biology. Key steps to deciphering the code are identifying the events in gene expression that are governed by individual "letters" and then defining a vocabulary of multiletter "words" and their meaning. Thr4 and Ser7 exert opposite effects on the fission yeast pho1 gene, expression of which is repressed under phosphate-replete conditions by transcription of an upstream flanking long noncoding RNA (lncRNA). Here we attribute the derepression of pho1 by a CTD-S7A mutation to precocious termination of lncRNA synthesis, an effect that is erased by mutations of cleavage-polyadenylation factor (CPF) subunits Ctf1, Ssu72, Ppn1, Swd22, and Dis2 and termination factor Rhn1. By contrast, a CTD-T4A mutation hyperrepresses pho1, as do CPF subunit and Rhn1 mutations, implying that T4A reduces lncRNA termination. Moreover, CTD-T4A is synthetically lethal with ppn1∆ and swd22∆, signifying that Thr4 and the Ppn1â¢Swd22 module play important, functionally redundant roles in promoting Pol2 termination. We find that Ppn1 and Swd22 become essential for viability when the CTD array is curtailed and that S7A overcomes the need for Ppn1â¢Swd22 in the short CTD context. Mutational synergies highlight redundant essential functions of (i) Ppn1â¢Swd22 and Rhn1, (ii) Ppn1â¢Swd22 and Ctf1, and (iii) Ssu72 and Dis2 phosphatases. CTD alleles Y1F, S2A, and T4A have overlapping synthetic lethalities with ppn1∆ and swd22∆, suggesting that Tyr1-Ser2-Thr4 form a three-letter CTD word that abets termination, with Rhn1 being a likely "reader" of this word.
Assuntos
Homeostase , Fosfatos/metabolismo , RNA Polimerase I/metabolismo , Schizosaccharomyces/metabolismo , Mutação , Fatores de Terminação de Peptídeos/genética , Fatores de Terminação de Peptídeos/metabolismo , Domínios Proteicos , RNA Polimerase I/química , Transdução de SinaisRESUMO
Expression of fission yeast glycerophosphate transporter Tgp1 is repressed in phosphate-rich medium and induced during phosphate starvation. Repression is enforced by transcription of the nc-tgp1 locus upstream of tgp1 to produce a long noncoding (lnc) RNA. Here we identify two essential elements of the nc-tgp1 promoter: a TATA box -30TATATATA-23 and a HomolD box -64CAGTCACA-57, mutations of which inactivate the nc-tgp1 promoter and de-repress the downstream tgp1 promoter under phosphate-replete conditions. The nc-tgp1 lncRNA poly(A) site maps to nucleotide +1636 of the transcription unit, which coincides with the binding site for Pho7 (1632TCGGACATTCAA1643), the transcription factor that drives tgp1 expression. Overlap between the lncRNA template and the tgp1 promoter points to transcriptional interference as the simplest basis for lncRNA repression. We identify a shorter RNA derived from the nc-tgp1 locus, polyadenylated at position +508, well upstream of the tgp1 promoter. Mutating the nc-tgp1-short RNA polyadenylation signal abolishes de-repression of the downstream tgp1 promoter elicited by Pol2 CTD Ser5Ala phospho-site mutation. Ser5 mutation favors utilization of the short RNA poly(A) site, thereby diminishing transcription of the lncRNA that interferes with the tgp1 promoter. Mutating the nc-tgp1-short RNA polyadenylation signal attenuates induction of the tgp1 promoter during phosphate starvation. Polyadenylation site choice governed by CTD Ser5 status adds a new level of lncRNA control of gene expression and reveals a new feature of the fission yeast CTD code.
Assuntos
Proteínas de Membrana Transportadoras/genética , RNA Polimerase II/genética , RNA Longo não Codificante/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Membrana Transportadoras/biossíntese , Mutação , Fosfatos/fisiologia , Poliadenilação , Regiões Promotoras Genéticas , RNA Fúngico/biossíntese , RNA Longo não Codificante/biossíntese , RNA Longo não Codificante/química , RNA Longo não Codificante/genética , Proteínas de Schizosaccharomyces pombe/biossíntese , Serina/genética , TATA Box , Sítio de Iniciação de TranscriçãoRESUMO
A seven-subunit Lsm2-8 protein ring assembles on the U-rich 3' end of the U6 snRNA. A structure-guided mutational analysis of the Saccharomyces cerevisiae Lsm2-8 ring affords new insights to structure-function relations and genetic interactions of the Lsm subunits. Alanine scanning of 39 amino acids comprising the RNA-binding sites or intersubunit interfaces of Lsm2, Lsm3, Lsm4, Lsm5, and Lsm8 identified only one instance of lethality (Lsm3-R69A) and one severe growth defect (Lsm2-R63A), both involving amino acids that bind the 3'-terminal UUU trinucleotide. All other Ala mutations were benign with respect to vegetative growth. Tests of 235 pairwise combinations of benign Lsm mutants identified six instances of inter-Lsm synthetic lethality and 45 cases of nonlethal synthetic growth defects. Thus, Lsm2-8 ring function is buffered by a network of internal genetic redundancies. A salient finding was that otherwise lethal single-gene deletions lsm2Δ, lsm3Δ, lsm4Δ, lsm5, and lsm8Δ were rescued by overexpression of U6 snRNA from a high-copy plasmid. Moreover, U6 overexpression rescued myriad lsmΔ lsmΔ double-deletions and lsmΔ lsmΔ lsmΔ triple-deletions. We find that U6 overexpression also rescues a lethal deletion of the U6 snRNP protein subunit Prp24 and that Prp24 overexpression bypasses the essentiality of the U6-associated Lsm subunits. Our results indicate that abetting U6 snRNA is the only essential function of the yeast Lsm2-8 proteins.
Assuntos
RNA Fúngico/genética , RNA Nuclear Pequeno/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sítios de Ligação , Mutação , Ligação Proteica , Splicing de RNA , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Deleção de SequênciaRESUMO
Pho7, a member of the Zn2Cys6 family of fungal transcription factors, is the key transcriptional activator underlying fission yeast phosphate homeostasis, a physiological response to phosphate starvation in which the pho1, pho84 and tgp1 genes are upregulated. Here, we delineated a minimized 61-amino-acid Pho7 DNA-binding domain (DBD) and determined the 1.7 Å crystal structure of the DBD at its target site in the tgp1 promoter. Two distinctive features of the Pho7 DBD are: it binds DNA as a monomer, unlike most other fungal zinc-cluster factors that bind as homodimers; and it makes extensive interactions with its asymmetric target sequence over a 14-bp footprint that entails hydrogen bonding to 13 individual bases within, and remote from, the CGG triplet typically recognized by other Zn2Cys6 DBDs. Base pair substitutions at Pho7 sites in the tgp1 and pho1 promoters highlight the importance of the 5'-CGG triplet for Pho7 binding in vitro and Pho7-dependent gene expression in vivo. We identify several DBD amino acids at which alanine substitution effaced or attenuated the pho1 phosphate starvation response and concordantly reduced Pho7 binding to a pho1 promoter site.