Publications by authors named "Beate Schwer"

83 Publications

Structure-function analysis of fission yeast cleavage and polyadenylation factor (CPF) subunit Ppn1 and its interactions with Dis2 and Swd22.

PLoS Genet 2021 Mar 12;17(3):e1009452. Epub 2021 Mar 12.

Molecular Biology Program, Sloan-Kettering Institute, New York, New York, United States of America.

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.
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http://dx.doi.org/10.1371/journal.pgen.1009452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7990198PMC
March 2021

Transcriptional profiling of fission yeast RNA polymerase II CTD mutants.

RNA 2021 Feb 12. Epub 2021 Feb 12.

Sloan Kettering Institute;

The carboxyl-terminal domain (CTD) of RNA polymerase II (Pol2) consists of tandem repeats of a consensus heptapeptide Y S P T S P S 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 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 , , and cells, and for and 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 P6•P6A and S5•S5A. De-repression of pho1 in P6•P6A and S5•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.
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http://dx.doi.org/10.1261/rna.078682.121DOI Listing
February 2021

Activity and substrate specificity of , , and Tpt1: essential tRNA splicing enzymes and potential anti-fungal targets.

RNA 2021 Jan 28. Epub 2021 Jan 28.

Sloan-Kettering Institute

The enzyme Tpt1 is an essential agent of fungal tRNA splicing that removes an internal RNA 2'-PO generated by fungal tRNA ligase. Tpt1 performs a two-step reaction in which: (i) the 2'-PO 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: , the agent of Valley Fever; and , which cause invasive, often fatal, infections in immunocompromised hosts; and , 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'-PO 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'-PO nucleotide with arabinose-2'-PO 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'-PO ribonucleotide as the principal determinant of fungal Tpt1 nucleic acid substrate specificity.
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http://dx.doi.org/10.1261/rna.078660.120DOI Listing
January 2021

A genetic screen for suppressors of hyper-repression of the fission yeast PHO regulon by Pol2 CTD mutation T4A implicates inositol 1-pyrophosphates as agonists of precocious lncRNA transcription termination.

Nucleic Acids Res 2020 11;48(19):10739-10752

Department of Microbiology and Immunology, Weill Cornell Medical College, New York, NY 10065, USA.

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.
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http://dx.doi.org/10.1093/nar/gkaa776DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641756PMC
November 2020

Inactivation of fission yeast Erh1 de-represses expression: evidence that Erh1 is a negative regulator of lncRNA termination.

RNA 2020 10 16;26(10):1334-1344. Epub 2020 Jun 16.

Molecular Biology Program, Sloan Kettering Institute, New York, New York 10065, USA.

Fission yeast Erh1 exists in a complex with RNA-binding protein Mmi1. Deletion of up-regulates the phosphate homeostasis gene , which is normally repressed by transcription in of a 5' flanking lncRNA. Here we present evidence that de-repression of by eΔ is achieved through precocious 3'-processing/termination of lncRNA synthesis, to wit: (i) Δ does not affect the activity of the or promoters per se; (ii) de-repression by Δ 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) Δ exerts an additive effect on 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 axis. In contrast, in Δ cells there is a reduction in mRNA and increase in the formation of a read-through transcript, consistent with Mmi1 being an agonist of termination. We envision that Erh1 acts as a brake on Mmi1's ability to promote CPF-dependent termination during lncRNA synthesis. Consistent with this idea, Δ de-repression of was eliminated by mutating the Mmi1-binding sites in the lncRNA.
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http://dx.doi.org/10.1261/rna.076463.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7491324PMC
October 2020

Genetic interactions and transcriptomics implicate fission yeast CTD prolyl isomerase Pin1 as an agent of RNA 3' processing and transcription termination that functions via its effects on CTD phosphatase Ssu72.

Nucleic Acids Res 2020 05;48(9):4811-4826

Dept. of Microbiology and Immunology, Weill Cornell Medical College, New York, NY 10065, USA.

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.
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http://dx.doi.org/10.1093/nar/gkaa212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7229847PMC
May 2020

Atomic structures of the RNA end-healing 5'-OH kinase and 2',3'-cyclic phosphodiesterase domains of fungal tRNA ligase: conformational switches in the kinase upon binding of the GTP phosphate donor.

Nucleic Acids Res 2019 12;47(22):11826-11838

Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.

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 β 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 GTP•Mg2+, IDP•PO4, and dGDP•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.
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http://dx.doi.org/10.1093/nar/gkz1049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7145591PMC
December 2019

Inositol pyrophosphates impact phosphate homeostasis via modulation of RNA 3' processing and transcription termination.

Nucleic Acids Res 2019 09;47(16):8452-8469

Department of Microbiology and Immunology, Weill Cornell Medical College, New York, NY 10065, USA.

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.
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http://dx.doi.org/10.1093/nar/gkz567DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6895273PMC
September 2019

Structure of Fission Yeast Transcription Factor Pho7 Bound to Promoter DNA and Effect of Pho7 Mutations on DNA Binding and Phosphate Homeostasis.

Mol Cell Biol 2019 07 13;39(13). Epub 2019 Jun 13.

Molecular Biology Program, Sloan-Kettering Institute, New York, New York, USA

Pho7 is the fission yeast ZnCys transcriptional factor that drives a response to phosphate starvation in which phosphate acquisition genes are upregulated. Here we report a crystal structure at 1.6-Å resolution of the Pho7 DNA-binding domain (DBD) bound at its target site 2 in the promoter (5'-TCGGAAATTAAAAA). Comparison to the previously reported structure of Pho7 DBD in complex with its binding site in the promoter (5'-TCGGACATTCAAAT) reveals shared determinants of target site specificity as well as variations in the protein-DNA interface that accommodate different promoter DNA sequences. Mutagenesis of Pho7 amino acids at the DNA interface identified nucleobase contacts at the periphery of the footprint that are essential for the induction of expression in response to phosphate starvation and for Pho7 binding to site 1 in the promoter.
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http://dx.doi.org/10.1128/MCB.00132-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6580706PMC
July 2019

Domain Requirements and Genetic Interactions of the Mud1 Subunit of the U1 snRNP.

G3 (Bethesda) 2019 01 9;9(1):145-151. Epub 2019 Jan 9.

Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065

Mud1 is an inessential 298-amino acid protein subunit of the U1 snRNP. Mud1 consists of N-terminal and C-terminal RRM domains (RRM1 and RRM2) separated by a linker domain. Synthetic lethal interactions of ∆ with deletions of inessential spliceosome components Nam8, Mud2, and Msl1, or missense mutations in the branchpoint-binding protein Msl5 enabled us to dissect genetically the domain requirements for Mud1 function. We find that the biological activities of Mud1 can be complemented by co-expressing separately the RRM1 (aa 1-127) and linker-RRM2 (aa 128-298) modules. Whereas RRM1 and RRM2 (aa 197-298) are inactive in all tests of functional complementation, the linker-RRM2 by itself partially complements a subset of synthetic lethal ∆ interactions. Linker segment aa 155 to 196 contains a nuclear localization signal rich in basic amino acids that is necessary for RRM2 activity in ∆ complementation. Alanine scanning mutagenesis indicates that none of the individual RRM1 amino acid contacts to U1 snRNA in the cryo-EM model of the yeast U1 snRNP is necessary for ∆ complementation activity.
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http://dx.doi.org/10.1534/g3.118.200781DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6325900PMC
January 2019

RNA polymerase II CTD interactome with 3' processing and termination factors in fission yeast and its impact on phosphate homeostasis.

Proc Natl Acad Sci U S A 2018 11 24;115(45):E10652-E10661. Epub 2018 Oct 24.

Department of Microbiology and Immunology, Weill Cornell Medical College, New York, NY 10065;

The carboxy-terminal domain (CTD) code encrypted within the YSPTSPS 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 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 by a CTD 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 mutation hyperrepresses , as do CPF subunit and Rhn1 mutations, implying that reduces lncRNA termination. Moreover, CTD is synthetically lethal with ∆ and ∆, signifying that Thr4 and the Ppn1•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 overcomes the need for Ppn1•Swd22 in the short CTD context. Mutational synergies highlight redundant essential functions of () Ppn1•Swd22 and Rhn1, () Ppn1•Swd22 and Ctf1, and () Ssu72 and Dis2 phosphatases. CTD alleles , , and have overlapping synthetic lethalities with ∆ and ∆, suggesting that Tyr1-Ser2-Thr4 form a three-letter CTD word that abets termination, with Rhn1 being a likely "reader" of this word.
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http://dx.doi.org/10.1073/pnas.1810711115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6233144PMC
November 2018

Distinctive structural basis for DNA recognition by the fission yeast Zn2Cys6 transcription factor Pho7 and its role in phosphate homeostasis.

Nucleic Acids Res 2018 11;46(21):11262-11273

Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.

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.
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http://dx.doi.org/10.1093/nar/gky827DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6265462PMC
November 2018

Defining essential elements and genetic interactions of the yeast Lsm2-8 ring and demonstration that essentiality of Lsm2-8 is bypassed via overexpression of U6 snRNA or the U6 snRNP subunit Prp24.

RNA 2018 06 3;24(6):853-864. Epub 2018 Apr 3.

Microbiology and Immunology Department, Weill Cornell Medical College, New York, New York 10065, USA.

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 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 Δ, Δ, Δ, , and Δ were rescued by overexpression of U6 snRNA from a high-copy plasmid. Moreover, U6 overexpression rescued myriad Δ Δ double-deletions and Δ Δ Δ 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.
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http://dx.doi.org/10.1261/rna.066175.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5959253PMC
June 2018

A long noncoding (lnc)RNA governs expression of the phosphate transporter Pho84 in fission yeast and has cascading effects on the flanking lncRNA and genes.

J Biol Chem 2018 03 2;293(12):4456-4467. Epub 2018 Feb 2.

the Department of Microbiology and Immunology, Weill Cornell Medical College, New York, New York 10065

The expression of the phosphate transporter Pho84 in fission yeast is repressed in phosphate-rich medium and induced during phosphate starvation. Two other phosphate-responsive genes in ( and ) had been shown to be repressed in by transcription of a long noncoding (lnc) RNA from the upstream flanking gene, but whether expression is regulated in this manner is unclear. Here, we show that repression of is enforced by transcription of the SPBC8E4.02c locus upstream of to produce a lncRNA that we name ( -epressive ranscript 2). We identify two essential elements of the promoter, a HomolD box and a TATA box, mutations of which inactivate the promoter and de-repress the downstream promoter under phosphate-replete conditions. We find that promoter inactivation also elicits a cascade effect on the adjacent downstream (lncRNA) and (acid phosphatase) genes, whereby increased transcription down-regulates lncRNA transcription and thereby de-represses Our results establish a unified model for the repressive arm of fission yeast phosphate homeostasis, in which transcription of , , and lncRNAs interferes with the promoters of the flanking , , and genes, respectively.
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http://dx.doi.org/10.1074/jbc.RA117.001352DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5868275PMC
March 2018

Poly(A) site choice and Pol2 CTD Serine-5 status govern lncRNA control of phosphate-responsive gene expression in fission yeast.

RNA 2018 02 9;24(2):237-250. Epub 2017 Nov 9.

Department of Microbiology and Immunology, Weill Cornell Medical College, New York, New York 10065, USA.

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 locus upstream of to produce a long noncoding (lnc) RNA. Here we identify two essential elements of the promoter: a TATA box TATATATA and a HomolD box CAGTCACA, mutations of which inactivate the promoter and de-repress the downstream promoter under phosphate-replete conditions. The lncRNA poly(A) site maps to nucleotide +1636 of the transcription unit, which coincides with the binding site for Pho7 (TCGGACATTCAA), the transcription factor that drives expression. Overlap between the lncRNA template and the promoter points to transcriptional interference as the simplest basis for lncRNA repression. We identify a shorter RNA derived from the locus, polyadenylated at position +508, well upstream of the promoter. Mutating the RNA polyadenylation signal abolishes de-repression of the downstream 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 promoter. Mutating the RNA polyadenylation signal attenuates induction of the 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.
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http://dx.doi.org/10.1261/rna.063966.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5769750PMC
February 2018

Defining the DNA Binding Site Recognized by the Fission Yeast ZnCys Transcription Factor Pho7 and Its Role in Phosphate Homeostasis.

mBio 2017 08 15;8(4). Epub 2017 Aug 15.

Molecular Biology Program, Sloan-Kettering Institute, New York, New York, USA

Fission yeast phosphate homeostasis entails transcriptional induction of genes encoding phosphate-mobilizing proteins under conditions of phosphate starvation. Transcription factor Pho7, a member of the ZnCys family of fungal transcription regulators, is the central player in the starvation response. The DNA binding sites in the promoters of phosphate-responsive genes have not been defined, nor have any structure-function relationships been established for the Pho7 protein. Here we narrow this knowledge gap by (i) delineating an autonomous DNA-binding domain (DBD) within Pho7 that includes the ZnCys module, (ii) deploying recombinant Pho7 DBD in DNase I footprinting and electrophoretic mobility shift assays (EMSAs) to map the Pho7 recognition sites in the promoters of the phosphate-regulated and genes to a 12-nucleotide sequence motif [5'-TCG(G/C)(A/T)xxTTxAA], (iii) independently identifying the same motif as a Pho7 recognition element via analysis of available genome-wide ChIP-seq data, (iv) affirming that mutations in the two Pho7 recognition sites in the promoter efface expression , and (v) establishing that the zinc-binding cysteines and a pair of conserved arginines in the DBD are essential for Pho7 activity Fungi respond to phosphate starvation by inducing the transcription of a set of phosphate acquisition genes that comprise a phosphate regulon. Pho7, a member of the ZnCys family of fungal transcription regulators, is the central player in the phosphate starvation response in fission yeast. The present study identifies a 12-nucleotide Pho7 DNA binding motif [5'-TCG(G/C)(A/T)xxTTxAA] in the promoters of phosphate-regulated genes, pinpoints DNA and protein features important for Pho7 binding to DNA, and correlates them with Pho7-dependent gene expression The results highlight distinctive properties of Pho7 vis-a-vis other fungal zinc binuclear cluster transcription factors as well as the divergent cast of transcription factors deployed for phosphate homeostasis in fission yeast versus budding yeast.
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http://dx.doi.org/10.1128/mBio.01218-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5559640PMC
August 2017

Will the circle be unbroken: specific mutations in the yeast Sm protein ring expose a requirement for assembly factor Brr1, a homolog of Gemin2.

RNA 2017 03 14;23(3):420-430. Epub 2016 Dec 14.

Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10065, USA

A seven-subunit Sm protein ring assembles around specific U-rich RNA segments of the U1, U2, U4, and U5 snRNPs that direct pre-mRNA splicing. Using human snRNP crystal structures to guide mutagenesis in , we gained new insights into structure-function relationships of the SmD1 and SmD2 subunits. Of 18 conserved amino acids comprising their RNA-binding sites or intersubunit interfaces, only Arg88 in SmD1 and Arg97 in SmD2 were essential for growth. Tests for genetic interactions with non-Sm splicing factors identified benign mutations of SmD1 (, , ) and SmD2 (, , , ) that were synthetically lethal with null alleles of U2 snRNP subunits Lea1 and/or Msl1. Tests of 264 pairwise combinations of SmD1 and SmD2 alleles with each other and with a collection of SmG, SmE, SmF, SmB, and SmD3 alleles revealed 92 instances of inter-Sm synthetic lethality. We leveraged the Sm mutant collection to illuminate the function of the yeast Sm assembly factor Brr1 and its relationship to the metazoan Sm assembly factor Gemin2. Mutations in the adjacent SmE (), SmF (, , ), SmD2 (, , , , ), and SmD1 (, ) subunits-but none in the SmG, SmD3, and SmB subunits-were synthetically lethal with Δ. Using complementation of Δ lethality in two Sm mutant backgrounds as an in vivo assay of Brr1 activity, we identified as essential an N-terminal segment of Brr1 (amino acids 24-47) corresponding to the Gemin2 α1 helix that interacts with SmF and a Brr1 C-terminal peptide (QKDLIE) that, in Gemin2, interacts with SmD2.
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http://dx.doi.org/10.1261/rna.059881.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5311505PMC
March 2017

Phospho-site mutants of the RNA Polymerase II C-terminal domain alter subtelomeric gene expression and chromatin modification state in fission yeast.

Nucleic Acids Res 2016 Nov 8;44(19):9180-9189. Epub 2016 Jul 8.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA

Eukaryotic gene expression requires that RNA Polymerase II (RNAP II) gain access to DNA in the context of chromatin. The C-terminal domain (CTD) of RNAP II recruits chromatin modifying enzymes to promoters, allowing for transcription initiation or repression. Specific CTD phosphorylation marks facilitate recruitment of chromatin modifiers, transcriptional regulators, and RNA processing factors during the transcription cycle. However, the readable code for recruiting such factors is still not fully defined and how CTD modifications affect related families of genes or regional gene expression is not well understood. Here, we examine the effects of manipulating the YSPTSPS heptapeptide repeat of the CTD of RNAP II in Schizosaccharomyces pombe by substituting non-phosphorylatable alanines for Ser2 and/or Ser7 and the phosphomimetic glutamic acid for Ser7. Global gene expression analyses were conducted using splicing-sensitive microarrays and validated via RT-qPCR. The CTD mutations did not affect pre-mRNA splicing or snRNA levels. Rather, the data revealed upregulation of subtelomeric genes and alteration of the repressive histone H3 lysine 9 methylation (H3K9me) landscape. The data further indicate that H3K9me and expression status are not fully correlated, suggestive of CTD-dependent subtelomeric repression mechansims that act independently of H3K9me levels.
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http://dx.doi.org/10.1093/nar/gkw603DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5100562PMC
November 2016

Mechanistic insights into the manganese-dependent phosphodiesterase activity of yeast Dbr1 with bis-p-nitrophenylphosphate and branched RNA substrates.

RNA 2016 12 7;22(12):1819-1827. Epub 2016 Oct 7.

Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10065, USA.

Saccharomyces cerevisiae Dbr1 is a manganese-dependent RNA debranching enzyme that cleaves the 2'-5' phosphodiester bond of the lariat introns formed during pre-mRNA splicing. Dbr1 is a member of the binuclear metallophosphoesterase enzyme superfamily. We showed previously via alanine scanning that RNA debranching in vivo and in vitro depends on conserved active site residues His13, Asp40, Asn85, His86, His179, His231, and His233. Here, by extending the alanine scan, we added Cys11 to the ensemble of essential active site components. We report that Dbr1 has a vigorous manganese-dependent phosphodiesterase activity with the non-RNA substrate bis-p-nitrophenylphosphate. Whereas RNA debranching requires His86, bis-p-nitrophenylphosphatase activity does not. We interpret these and other structure-activity relations reported here in light of the crystal structures of Entamoeba Dbr1 and other homologous binuclear metallophosphodiesterases. Our results suggest that (i) Dbr1 adheres to the two-metal mechanism of the enzyme superfamily, but is distinguished by its reliance on a Cys11-Xaa-His13 motif to engage one of the catalytic metals instead of the Asp-Xaa-His element typical of other clades within the superfamily; (ii) His86 is a general acid catalyst that protonates the O2' leaving group of the RNA 2'-5' phosphodiester; and (iii) the favorable pK of p-nitrophenol elides the strict need for a general acid during hydrolysis of bis-p-nitrophenylphosphate. The Dbr1 bis-p-nitrophenylphosphatase activity is well suited for high-throughput screening for inhibitors of debranching.
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http://dx.doi.org/10.1261/rna.058552.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5113202PMC
December 2016

Characterization of the tRNA ligases of pathogenic fungi Aspergillus fumigatus and Coccidioides immitis.

RNA 2016 10 4;22(10):1500-9. Epub 2016 Aug 4.

Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10065, USA.

Yeast tRNA ligase (Trl1) is an essential trifunctional enzyme that repairs RNA breaks with 2',3'-cyclic-PO4 and 5'-OH ends. Trl1 is composed of C-terminal cyclic phosphodiesterase and central polynucleotide kinase domains that heal the broken ends to generate the 3'-OH, 2'-PO4, and 5'-PO4 termini required for sealing by an N-terminal ligase domain. Trl1 enzymes are found in all human fungal pathogens and they are promising targets for antifungal drug discovery because: (i) their domain structures and biochemical mechanisms are unique compared to the mammalian RtcB-type tRNA splicing enzyme; and (ii) there are no obvious homologs of the Trl1 ligase domain in mammalian proteomes. Here we characterize the tRNA ligases of two human fungal pathogens: Coccidioides immitis and Aspergillus fumigatus The biological activity of CimTrl1 and AfuTrl1 was verified by showing that their expression complements a Saccharomyces cerevisiae trl1Δ mutant. Purified recombinant AfuTrl1 and CimTrl1 proteins were catalytically active in joining 2',3'-cyclic-PO4 and 5'-OH ends in vitro, either as full-length proteins or as a mixture of separately produced healing and sealing domains. The biochemical properties of CimTrl1 and AfuTrl1 are similar to those of budding yeast Trl1, particularly with respect to their preferential use of GTP as the phosphate donor for the polynucleotide kinase reaction. Our findings provide genetic and biochemical tools to screen for inhibitors of tRNA ligases from pathogenic fungi.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5029449PMC
http://dx.doi.org/10.1261/rna.057455.116DOI Listing
October 2016

Structure-function analysis and genetic interactions of the SmG, SmE, and SmF subunits of the yeast Sm protein ring.

RNA 2016 09 14;22(9):1320-8. Epub 2016 Jul 14.

Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10065, USA.

A seven-subunit Sm protein ring forms a core scaffold of the U1, U2, U4, and U5 snRNPs that direct pre-mRNA splicing. Using human snRNP structures to guide mutagenesis in Saccharomyces cerevisiae, we gained new insights into structure-function relationships of the SmG, SmE, and SmF subunits. An alanine scan of 19 conserved amino acids of these three proteins, comprising the Sm RNA binding sites or inter-subunit interfaces, revealed that, with the exception of Arg74 in SmF, none are essential for yeast growth. Yet, for SmG, SmE, and SmF, as for many components of the yeast spliceosome, the effects of perturbing protein-RNA and protein-protein interactions are masked by built-in functional redundancies of the splicing machine. For example, tests for genetic interactions with non-Sm splicing factors showed that many benign mutations of SmG, SmE, and SmF (and of SmB and SmD3) were synthetically lethal with null alleles of U2 snRNP subunits Lea1 and Msl1. Tests of pairwise combinations of SmG, SmE, SmF, SmB, and SmD3 alleles highlighted the inherent redundancies within the Sm ring, whereby simultaneous mutations of the RNA binding sites of any two of the Sm subunits are lethal. Our results suggest that six intact RNA binding sites in the Sm ring suffice for function but five sites may not.
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http://dx.doi.org/10.1261/rna.057448.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4986888PMC
September 2016

Structure-function analysis and genetic interactions of the Luc7 subunit of the Saccharomyces cerevisiae U1 snRNP.

RNA 2016 09 27;22(9):1302-10. Epub 2016 Jun 27.

Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10065, USA.

Luc7 is an essential 261-amino acid protein subunit of the Saccharomyces cerevisiae U1 snRNP. To establish structure-function relations for yeast Luc7, we conducted an in vivo mutational analysis entailing N- and C-terminal truncations and alanine scanning of phylogenetically conserved amino acids, including two putative zinc finger motifs, ZnF1 and ZnF2, and charged amino acids within the ZnF2 module. We identify Luc7-(31-246) as a minimal functional protein and demonstrate that whereas mutations of the CCHH ZnF2 motif are lethal, mutations of the ZnF1 CCCH motif and the charged residues of the ZnF2 modules are not. Though dispensable for vegetative growth in an otherwise wild-type background, the N-terminal 18-amino acid segment of Luc7 plays an important role in U1 snRNP function, evinced by our findings that its deletion (i) impaired the splicing of SUS1 pre-mRNA; (ii) was synthetically lethal absent other U1 snRNP constituents (Mud1, Nam8, the TMG cap, the C terminus of Snp1), absent the Mud2 subunit of the Msl5•Mud2 branchpoint binding complex, and when the m(7)G cap-binding site of Cbc2 was debilitated; and (iii) bypassed the need for the essential DEAD-box ATPase Prp28. Similar phenotypes were noted for ZnF1 mutations C45A, C53A, and C68A and ZnF2 domain mutations D214A, R215A, R216A, and D219A These findings highlight the contributions of the Luc7 N-terminal peptide, the ZnF1 motif, and the ZnF2 module in stabilizing the interactions of the U1 snRNP with the pre-mRNA 5' splice site and promoting the splicing of a yeast pre-mRNA, SUS1, that has a nonconsensus 5' splice site.
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http://dx.doi.org/10.1261/rna.056911.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4986886PMC
September 2016

Transcription of lncRNA prt, clustered prt RNA sites for Mmi1 binding, and RNA polymerase II CTD phospho-sites govern the repression of pho1 gene expression under phosphate-replete conditions in fission yeast.

RNA 2016 07 10;22(7):1011-25. Epub 2016 May 10.

Department of Microbiology and Immunology, Weill Cornell Medical College, New York, New York 10065, USA.

Expression of fission yeast Pho1 acid phosphatase is repressed during growth in phosphate-rich medium. Repression is mediated by transcription of the prt locus upstream of pho1 to produce a long noncoding (lnc) prt RNA. Repression is also governed by RNA polymerase II CTD phosphorylation status, whereby inability to place a Ser7-PO4 mark (as in S7A) derepresses Pho1 expression, and inability to place a Thr4-PO4 mark (as in T4A) hyper-represses Pho1 in phosphate replete cells. Here we find that basal pho1 expression from the prt-pho1 locus is inversely correlated with the activity of the prt promoter, which resides in a 110-nucleotide DNA segment preceding the prt transcription start site. CTD mutations S7A and T4A had no effect on the activity of the prt promoter or the pho1 promoter, suggesting that S7A and T4A affect post-initiation events in prt lncRNA synthesis that make it less and more repressive of pho1, respectively. prt lncRNA contains clusters of DSR (determinant of selective removal) sequences recognized by the YTH-domain-containing protein Mmi1. Altering the nucleobase sequence of two DSR clusters in the prt lncRNA caused hyper-repression of pho1 in phosphate replete cells, concomitant with increased levels of the prt transcript. The isolated Mmi1 YTH domain binds to RNAs with single or tandem DSR elements, to the latter in a noncooperative fashion. We report the 1.75 Å crystal structure of the Mmi1 YTH domain and provide evidence that Mmi1 recognizes DSR RNA via a binding mode distinct from that of structurally homologous YTH proteins that recognize m(6)A-modified RNA.
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http://dx.doi.org/10.1261/rna.056515.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4911910PMC
July 2016

Functional interaction of Rpb1 and Spt5 C-terminal domains in co-transcriptional histone modification.

Nucleic Acids Res 2015 Nov 14;43(20):9766-75. Epub 2015 Aug 14.

Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, H3G 1Y6, Canada

Transcription by RNA polymerase II (RNAPII) is accompanied by a conserved pattern of histone modifications that plays important roles in regulating gene expression. The establishment of this pattern requires phosphorylation of both Rpb1 (the largest RNAPII subunit) and the elongation factor Spt5 on their respective C-terminal domains (CTDs). Here we interrogated the roles of individual Rpb1 and Spt5 CTD phospho-sites in directing co-transcriptional histone modifications in the fission yeast Schizosaccharomyces pombe. Steady-state levels of methylation at histone H3 lysines 4 (H3K4me) and 36 (H3K36me) were sensitive to multiple mutations of the Rpb1 CTD repeat motif (Y1S2P3T4S5P6S7). Ablation of the Spt5 CTD phospho-site Thr1 reduced H3K4me levels but had minimal effects on H3K36me. Nonetheless, Spt5 CTD mutations potentiated the effects of Rpb1 CTD mutations on H3K36me, suggesting overlapping functions. Phosphorylation of Rpb1 Ser2 by the Cdk12 orthologue Lsk1 positively regulated H3K36me but negatively regulated H3K4me. H3K36me and histone H2B monoubiquitylation required Rpb1 Ser5 but were maintained upon inactivation of Mcs6/Cdk7, the major kinase for Rpb1 Ser5 in vivo, implicating another Ser5 kinase in these regulatory pathways. Our results elaborate the CTD 'code' for co-transcriptional histone modifications.
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http://dx.doi.org/10.1093/nar/gkv837DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4787787PMC
November 2015

RNA polymerase II CTD phospho-sites Ser5 and Ser7 govern phosphate homeostasis in fission yeast.

RNA 2015 Oct 11;21(10):1770-80. Epub 2015 Aug 11.

Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10065, USA.

Phosphorylation of the tandem YSPTSPS repeats of the RNA polymerase II CTD inscribes an informational code that orchestrates eukaryal mRNA synthesis. Here we interrogate the role of the CTD in phosphate homeostasis in fission yeast. Expression of Pho1 acid phosphatase, which is repressed during growth in phosphate-rich medium and induced by phosphate starvation, is governed strongly by CTD phosphorylation status, but not by CTD repeat length. Inability to place a Ser7-PO4 mark (as in S7A) results in constitutive derepression of Pho1 expression in phosphate-replete medium. In contrast, indelible installation of a Ser7-PO4 mimetic (as in S7E) hyper-represses Pho1 in phosphate-replete cells and inhibits Pho1 induction during starvation. Pho1 phosphatase is derepressed by ablation of the CTD Ser5-PO4 mark, achieved either by mutating Ser5 in all consensus heptads to alanine, or replacing all Pro6 residues with alanine. We find that Ser5 status is a tunable determinant of Pho1 regulation, i.e., serial decrements in the number of consensus Ser5 heptads from seven to two elicits a progressive increase in Pho1 expression in phosphate-replete medium. Pho1 is also derepressed by hypomorphic mutations of the CTD kinase Cdk9. Inactivation of the CTD phosphatase Ssu72 attenuates Pho1 induction in wild-type cells and blocks Pho1 derepression in S7A cells. These experiments implicate Ser5, Pro6, and Ser7 as component letters of a CTD coding "word" that transduces a repressive transcriptional signal via serine phosphorylation.
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http://dx.doi.org/10.1261/rna.052555.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4574753PMC
October 2015

Two Routes to Genetic Suppression of RNA Trimethylguanosine Cap Deficiency via C-Terminal Truncation of U1 snRNP Subunit Snp1 or Overexpression of RNA Polymerase Subunit Rpo26.

G3 (Bethesda) 2015 Apr 24;5(7):1361-70. Epub 2015 Apr 24.

Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10065

The trimethylguanosine (TMG) caps of small nuclear (sn) RNAs are synthesized by the enzyme Tgs1 via sequential methyl additions to the N2 atom of the m(7)G cap. Whereas TMG caps are inessential for Saccharomyces cerevisiae vegetative growth at 25° to 37°, tgs1∆ cells that lack TMG caps fail to thrive at 18°. The cold-sensitive defect correlates with ectopic stoichiometric association of nuclear cap-binding complex (CBC) with the residual m(7)G cap of the U1 snRNA and is suppressed fully by Cbc2 mutations that weaken cap binding. Here, we show that normal growth of tgs1∆ cells at 18° is also restored by a C-terminal deletion of 77 amino acids from the Snp1 subunit of yeast U1 snRNP. These results underscore the U1 snRNP as a focal point for TMG cap function in vivo. Casting a broader net, we conducted a dosage suppressor screen for genes that allowed survival of tgs1∆ cells at 18°. We thereby recovered RPO26 (encoding a shared subunit of all three nuclear RNA polymerases) and RPO31 (encoding the largest subunit of RNA polymerase III) as moderate and weak suppressors of tgs1∆ cold sensitivity, respectively. A structure-guided mutagenesis of Rpo26, using rpo26∆ complementation and tgs1∆ suppression as activity readouts, defined Rpo26-(78-155) as a minimized functional domain. Alanine scanning identified Glu89, Glu124, Arg135, and Arg136 as essential for rpo26∆ complementation. The E124A and R135A alleles retained tgs1∆ suppressor activity, thereby establishing a separation-of-function. These results illuminate the structure activity profile of an essential RNA polymerase component.
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http://dx.doi.org/10.1534/g3.115.016675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4502370PMC
April 2015

Structure-function analysis and genetic interactions of the Yhc1, SmD3, SmB, and Snp1 subunits of yeast U1 snRNP and genetic interactions of SmD3 with U2 snRNP subunit Lea1.

RNA 2015 Jun 20;21(6):1173-86. Epub 2015 Apr 20.

Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10065, USA.

Yhc1 and U1-C are essential subunits of the yeast and human U1 snRNP, respectively, that stabilize the duplex formed by U1 snRNA at the pre-mRNA 5' splice site (5'SS). Mutational analysis of Yhc1, guided by the human U1 snRNP crystal structure, highlighted the importance of Val20 and Ser19 at the RNA interface. Though benign on its own, V20A was lethal in the absence of branchpoint-binding complex subunit Mud2 and caused a severe growth defect in the absence of U1 subunit Nam8. S19A caused a severe defect with mud2▵. Essential DEAD-box ATPase Prp28 was bypassed by mutations of Yhc1 Val20 and Ser19, consistent with destabilization of U1•5'SS interaction. We extended the genetic analysis to SmD3, which interacts with U1-C/Yhc1 in U1 snRNP, and to SmB, its neighbor in the Sm ring. Whereas mutations of the interface of SmD3, SmB, and U1-C/Yhc1 with U1-70K/Snp1, or deletion of the interacting Snp1 N-terminal peptide, had no growth effect, they elicited synthetic defects in the absence of U1 subunit Mud1. Mutagenesis of the RNA-binding triad of SmD3 (Ser-Asn-Arg) and SmB (His-Asn-Arg) provided insights to built-in redundancies of the Sm ring, whereby no individual side-chain was essential, but simultaneous mutations of Asn or Arg residues in SmD3 and SmB were lethal. Asn-to-Ala mutations SmB and SmD3 caused synthetic defects in the absence of Mud1 or Mud2. All three RNA site mutations of SmD3 were lethal in cells lacking the U2 snRNP subunit Lea1. Benign C-terminal truncations of SmD3 were dead in the absence of Mud2 or Lea1 and barely viable in the absence of Nam8 or Mud1. In contrast, SMD3-E35A uniquely suppressed the temperature-sensitivity of lea1▵.
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http://dx.doi.org/10.1261/rna.050583.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4436669PMC
June 2015

Genetic and structural analysis of the essential fission yeast RNA polymerase II CTD phosphatase Fcp1.

RNA 2015 Jun 16;21(6):1135-46. Epub 2015 Apr 16.

Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10065, USA.

Protein phosphatases regulate mRNA synthesis and processing by remodeling the carboxy-terminal domain (CTD) of RNA polymerase II (Pol2) to dynamically inscribe a Pol2 CTD code. Fission yeast Fcp1 (SpFcp1) is an essential 723-amino acid CTD phosphatase that preferentially hydrolyzes Ser2-PO4 of the YS(2)PTSPS repeat. The SpFcp1 catalytic domain (aa 140-580) is composed of a DxDxT acyl-phosphatase module (FCPH) and a BRCT module. Here we conducted a genetic analysis of SpFcp1, which shows that (i) phosphatase catalytic activity is required for vegetative growth of fission yeast; (ii) the flanking amino-terminal domain (aa 1-139) and its putative metal-binding motif C(99)H(101)Cys(109)C(112) are essential; (iii) the carboxy-terminal domain (aa 581-723) is dispensable; (iv) a structurally disordered internal segment of the FCPH domain (aa 330-393) is dispensable; (v) lethal SpFcp1 mutations R271A and R299A are rescued by shortening the Pol2 CTD repeat array; and (vi) CTD Ser2-PO4 is not the only essential target of SpFcp1 in vivo. Recent studies highlight a second CTD code involving threonine phosphorylation of a repeat motif in transcription elongation factor Spt5. We find that Fcp1 can dephosphorylate Thr1-PO4 of the fission yeast Spt5 CTD nonamer repeat T(1)PAWNSGSK. We identify Arg271 as a governor of Pol2 versus Spt5 CTD substrate preference. Our findings implicate Fcp1 as a versatile sculptor of both the Pol2 and Spt5 CTD codes. Finally, we report a new 1.45 Å crystal structure of SpFcp1 with Mg(2+) and AlF3 that mimics an associative phosphorane transition state of the enzyme-aspartyl-phosphate hydrolysis reaction.
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http://dx.doi.org/10.1261/rna.050286.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4436666PMC
June 2015

Structural basis for recognition of intron branchpoint RNA by yeast Msl5 and selective effects of interfacial mutations on splicing of yeast pre-mRNAs.

RNA 2015 Mar 13;21(3):401-14. Epub 2015 Jan 13.

Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10065, USA

Saccharomyces cerevisiae Msl5 orchestrates spliceosome assembly by binding the intron branchpoint sequence 5'-UACUAAC and, with its heterodimer partner protein Mud2, establishing cross intron-bridging interactions with the U1 snRNP at the 5' splice site. Here we define the central Msl5 KH-QUA2 domain as sufficient for branchpoint RNA recognition. The 1.8 Å crystal structure of Msl5-(KH-QUA2) bound to the branchpoint highlights an extensive network of direct and water-mediated protein-RNA and intra-RNA atomic contacts at the interface that illuminate how Msl5 recognizes each nucleobase of the UACUAAC element. The Msl5 structure rationalizes a large body of mutational data and inspires new functional studies herein, which reveal how perturbations of the Msl5·RNA interface impede the splicing of specific yeast pre-mRNAs. We also identify interfacial mutations in Msl5 that bypass the essentiality of Sub2, a DExD-box ATPase implicated in displacing Msl5 from the branchpoint in exchange for the U2 snRNP. These studies establish an atomic resolution framework for understanding splice site selection and early spliceosome dynamics.
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http://dx.doi.org/10.1261/rna.048942.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4338336PMC
March 2015

Fission yeast RNA triphosphatase reads an Spt5 CTD code.

RNA 2015 Jan 20;21(1):113-23. Epub 2014 Nov 20.

Structural Biology Program, Sloan-Kettering Institute, New York, New York 10065, USA Howard Hughes Medical Institute, Structural Biology Program, Sloan-Kettering Institute, New York, New York 10065, USA

mRNA capping enzymes are directed to nascent RNA polymerase II (Pol2) transcripts via interactions with the carboxy-terminal domains (CTDs) of Pol2 and transcription elongation factor Spt5. Fission yeast RNA triphosphatase binds to the Spt5 CTD, comprising a tandem repeat of nonapeptide motif TPAWNSGSK. Here we report the crystal structure of a Pct1·Spt5-CTD complex, which revealed two CTD docking sites on the Pct1 homodimer that engage TPAWN segments of the motif. Each Spt5 CTD interface, composed of elements from both subunits of the homodimer, is dominated by van der Waals contacts from Pct1 to the tryptophan of the CTD. The bound CTD adopts a distinctive conformation in which the peptide backbone makes a tight U-turn so that the proline stacks over the tryptophan. We show that Pct1 binding to Spt5 CTD is antagonized by threonine phosphorylation. Our results fortify an emerging concept of an "Spt5 CTD code" in which (i) the Spt5 CTD is structurally plastic and can adopt different conformations that are templated by particular cellular Spt5 CTD receptor proteins; and (ii) threonine phosphorylation of the Spt5 CTD repeat inscribes a binary on-off switch that is read by diverse CTD receptors, each in its own distinctive manner.
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http://dx.doi.org/10.1261/rna.048181.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4274631PMC
January 2015