Lauren T. Neves

In eukaryotes, a dynamic ribonucleic protein machine known as the spliceosome catalyzes the removal of introns from premessenger RNA (pre-mRNA). Recent studies show the processes of RNA synthesis and RNA processing to be spatio–temporally coordinated, indicating that RNA splicing takes place in the context of chromatin. H2A.Z is a highly conserved histone variant of the canonical histone H2A. In Saccharomyces cerevisiae , H2A.Z is deposited into chromatin by the SWR-C complex, is found near the 5′ ends of protein-coding genes, and has been implicated in transcription regulation. Here we show that splicing of intron-containing genes in cells lacking H2A.Z is impaired, particularly under suboptimal splicing conditions. Cells lacking H2A.Z are especially dependent on a functional U2 snRNP (small nuclear RNA [snRNA] plus associated proteins), as H2A.Z shows extensive genetic interactions with U2 snRNP-associated proteins, and RNA sequencing (RNA-seq) reveals that introns with nonconsensus branch points are particularly sensitive to H2A.Z loss. Consistently, H2A.Z promotes efficient spliceosomal rearrangements involving the U2 snRNP, as H2A.Z loss results in persistent U2 snRNP association and decreased recruitment of downstream snRNPs to nascent RNA. H2A.Z impairs transcription elongation, suggesting that spliceosome rearrangements are tied to H2A.Z's role in elongation. Depletion of disassembly factor Prp43 suppresses H2A.Z-mediated splice defects, indicating that, in the absence of H2A.Z, stalled spliceosomes are disassembled, and unspliced RNAs are released. Together, these data demonstrate that H2A.Z is required for efficient pre-mRNA splicing and indicate a role for H2A.Z in coordinating the kinetics of transcription elongation and splicing.

Despite its relatively streamlined genome, there are many important examples of regulated RNA splicing in Saccharomyces cerevisiae. Here, we report a role for the chromatin remodeler SWI/SNF in respiration, partially via the regulation of splicing. We find that a nutrient-dependent decrease in Snf2 leads to an increase in splicing of the PTC7 transcript. The spliced PTC7 transcript encodes a mitochondrial phosphatase regulator of biosynthesis of coenzyme Q6 (ubiquinone or CoQ6) and a mitochondrial redox-active lipid essential for electron and proton transport in respiration. Increased splicing of PTC7 increases CoQ6 levels. The increase in PTC7 splicing occurs at least in part due to down-regulation of ribosomal protein gene expression, leading to the redistribution of spliceosomes from this abundant class of intron-containing RNAs to otherwise poorly spliced transcripts. In contrast, a protein encoded by the nonspliced isoform of PTC7 represses CoQ6 biosynthesis. Taken together, these findings uncover a link between Snf2 expression and the splicing of PTC7 and establish a previously unknown role for the SWI/SNF complex in the transition of yeast cells from fermentative to respiratory modes of metabolism. Despite its relatively streamlined genome, there are many important examples of regulated RNA splicing in Saccharomyces cerevisiae. Here, we report a role for the chromatin remodeler SWI/SNF in respiration, partially via the regulation of splicing. We find that a nutrient-dependent decrease in Snf2 leads to an increase in splicing of the PTC7 transcript. The spliced PTC7 transcript encodes a mitochondrial phosphatase regulator of biosynthesis of coenzyme Q6 (ubiquinone or CoQ6) and a mitochondrial redox-active lipid essential for electron and proton transport in respiration. Increased splicing of PTC7 increases CoQ6 levels. The increase in PTC7 splicing occurs at least in part due to down-regulation of ribosomal protein gene expression, leading to the redistribution of spliceosomes from this abundant class of intron-containing RNAs to otherwise poorly spliced transcripts. In contrast, a protein encoded by the nonspliced isoform of PTC7 represses CoQ6 biosynthesis. Taken together, these findings uncover a link between Snf2 expression and the splicing of PTC7 and establish a previously unknown role for the SWI/SNF complex in the transition of yeast cells from fermentative to respiratory modes of metabolism. Similar to other eukaryotic genomes, genes in Saccharomyces cerevisiae may be interrupted by non-coding sequences, called introns. Introns are removed from the pre-mRNA through the action of the spliceosome, a macromolecular machine composed of five small nuclear ribonucleoproteins. The spliceosome recognizes consensus sequence signals on the pre-mRNA, termed splice sites, by which it subsequently binds to the intron and catalyzes its removal via two transesterification reactions (1.Naftelberg S. Schor I.E. Ast G. Kornblihtt A.R. Regulation of alternative splicing through coupling with transcription and chromatin structure.Annu. Rev. Biochem. 2015; 84: 165-198Crossref PubMed Scopus (279) Google Scholar). Pre-mRNA splicing is critical for accurate gene expression in all eukaryotes, and there is significant evidence that alterations in microenvironments, such as changes in the chromatin state or chromatin-modifying factors, can affect splicing outcomes (1.Naftelberg S. Schor I.E. Ast G. Kornblihtt A.R. Regulation of alternative splicing through coupling with transcription and chromatin structure.Annu. Rev. Biochem. 2015; 84: 165-198Crossref PubMed Scopus (279) Google Scholar). However, the mechanisms for how chromatin and chromatin factors influence splicing are not completely understood. Although the genome of S. cerevisiae contains a smaller number of introns than metazoan genomes, there are, nonetheless, numerous examples of intron-dependent gene regulation (2.Johnson T.L. Vilardell J. Regulated pre-mRNA splicing: the ghostwriter of the eukaryotic genome.Biochim. Biophys. Acta. 2012; 1819: 538-545Crossref PubMed Scopus (22) Google Scholar). The largest functional class of intron-containing genes (ICGs) 4The abbreviations used are: ICG, intron-containing gene; RPG, ribosomal protein gene; ns, non-spliced; s, spliced; CoQ, coenzyme Q; DMQ6, 5-demethoxy-Q6; 4HB, 4-hydroxybenzoic acid; HHB, 3-hexaprenyl-4-hydroxybenzoic acid; qPCR, quantitative PCR; TOR, target of rapamycin. 4The abbreviations used are: ICG, intron-containing gene; RPG, ribosomal protein gene; ns, non-spliced; s, spliced; CoQ, coenzyme Q; DMQ6, 5-demethoxy-Q6; 4HB, 4-hydroxybenzoic acid; HHB, 3-hexaprenyl-4-hydroxybenzoic acid; qPCR, quantitative PCR; TOR, target of rapamycin. in budding yeast is ribosomal protein genes (RPGs) that encode the protein components of the ribosome. Therefore, the energy-intensive process of translation is under the heavy regulatory control of the spliceosome, such that splicing of RPGs can be finely tuned to the cells' environmental conditions and to nutrient availability (3.Pleiss J.A. Whitworth G.B. Bergkessel M. Guthrie C. Rapid, transcript-specific changes in splicing in response to environmental stress.Mol. Cell. 2007; 27: 928-937Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Interestingly, this enrichment of introns within RPGs impacts the splicing of, as well as provides an opportunity for the regulation of, other ICGs within the yeast genome. About a third of yeast introns occur in RPGs, and the high transcription of these genes that of the intron by the spliceosome is from this functional class of genes M. of intron-containing genes the of yeast PubMed Scopus Google Scholar). the of introns to spliceosomes from other splice expression the splicing of splice is the process of yeast conditions of a number of ICGs are splice and are poorly by the spliceosome and However, the down-regulation of RPGs availability of the previously of spliceosomes leads to splicing of introns in S. M. between for the splicing regulation of Cell. Full Text Full Text PDF PubMed Scopus Google S. S. A.R. T.L. The chromatin complex splicing of in Saccharomyces PubMed Scopus Google Scholar). are other important examples of regulation in S. ICGs with splice J. T.L. regulation of expression and is for in response to Cell. Full Text Full Text PDF PubMed Scopus Google T.L. of the Saccharomyces cerevisiae gene to its alternative PubMed Scopus Google Scholar). such gene is which encodes a protein phosphatase M. The gene encodes a protein phosphatase and the gene in budding PubMed Scopus Google Scholar). The intron within PTC7 is it contains a its splicing relatively under The PTC7 intron a and is The of the PTC7 RNA encodes a protein that contains a the is otherwise to the protein isoform from the spliced PTC7 RNA The of the PTC7 intron is yeast for and protein C. splicing of cerevisiae protein PubMed Scopus Google Scholar). to the nuclear is within C. splicing of cerevisiae protein PubMed Scopus Google Scholar). in regulation of coenzyme termed or biosynthesis via its phosphatase S. M. C. The phosphatase coenzyme biosynthesis by the in Full Text Full Text PDF PubMed Scopus Google mitochondrial to Full Text Full Text PDF PubMed Scopus Google Scholar). However, mechanisms of regulation of and the role of the isoform is a redox-active lipid composed of a and a and is for mitochondrial electron The of the is and S. cerevisiae with and The role of in the mitochondrial is to the from complex and complex and to complex other such as and on as an electron M. J. G. and of coenzyme Biophys. Acta. PubMed Scopus Google Scholar). is in all it may as a and are to in biosynthesis M. C. M. of coenzyme PubMed Scopus Google M. of coenzyme PubMed Scopus Google of coenzyme in 2007; PubMed Scopus Google S. C. The regulation of coenzyme biosynthesis in eukaryotic all that yeast can PubMed Scopus (22) Google Scholar). are for the biosynthesis of CoQ6 in S. cerevisiae and C. J.A. of a coenzyme protein in the in Saccharomyces 2015; Full Text Full Text PDF PubMed Scopus Google Scholar). of the for the biosynthesis of CoQ6 in a complex the a complex that is with the mitochondrial on the C. J.A. of a coenzyme protein in the in Saccharomyces 2015; Full Text Full Text PDF PubMed Scopus Google Scholar). to to the it is to the state of S. M. C. The phosphatase coenzyme biosynthesis by the in Full Text Full Text PDF PubMed Scopus Google influence mitochondrial respiratory mitochondrial to Full Text Full Text PDF PubMed Scopus Google Scholar). In the is to at least in the state of the which its catalyzes the of the in the biosynthesis of CoQ6 in yeast S. G. C. an of coenzyme to in Saccharomyces cerevisiae and Full Text Full Text PDF PubMed Scopus Google S. M. C. of a control in yeast coenzyme Q6 PubMed Scopus Google Scholar). The SWI/SNF complex by the to in the or of from the a Snf2 to regulation by the yeast SWI/SNF PubMed Scopus Google M. of chromatin by the PubMed Scopus Google Scholar). The of SWI/SNF is to conditions of and the complex is for transcription of a number of response genes M. M. S. J. on genes is by PubMed Scopus Google M. M. J. and of Full Text Full Text PDF PubMed Scopus Google Scholar). We previously that of Snf2 in response to nutrient We that the in Snf2 leads to changes in of splicing outcomes S. S. A.R. T.L. The chromatin complex splicing of in Saccharomyces PubMed Scopus Google Scholar). Here, we that changes in of Snf2 the CoQ6 in S. cerevisiae. we that of Snf2 the of and in yeast and increases the of and of is due to down-regulation of and an increase in the of we find that the Snf2 protein is under conditions and nutrient and with a increase in the splicing of this leads to CoQ6 in for the transition from a fermentative of to a respiratory we that the two on the CoQ6 which may in the the of on CoQ6 S. M. C. The phosphatase coenzyme biosynthesis by the in Full Text Full Text PDF PubMed Scopus Google mitochondrial to Full Text Full Text PDF PubMed Scopus Google Scholar). Snf2 is in response to it is for on that control of Snf2 is for the transition from to respiration. RNA for yeast the of the SWI/SNF complex number an increase in splicing of a number of introns S. S. A.R. T.L. The chromatin complex splicing of in Saccharomyces PubMed Scopus Google Scholar). the in splicing of Snf2 is by via a previously with down-regulation of expression J. G. expression in Saccharomyces PubMed Scopus Google Scholar). The two largest in splicing are by of unknown and a previously mitochondrial phosphatase that contains all of the protein previously to a role in CoQ6 biosynthesis in yeast S. M. C. The phosphatase coenzyme biosynthesis by the in Full Text Full Text PDF PubMed Scopus Google increase in splicing of PTC7 RNA by In the from the and by not previously that splicing of poorly introns can be by expression of RPGs S. M. between for the splicing regulation of Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). we that of Snf2 down-regulation of RPGs and in splicing of a number of introns S. S. A.R. T.L. The chromatin complex splicing of in Saccharomyces PubMed Scopus Google Scholar). down-regulation in the of Snf2 by expression of and two intron-containing RPGs, is in yeast with The PTC7 transcript two protein from the nonspliced and from the spliced The spliced isoform to the the nonspliced isoform to to the nuclear C. splicing of cerevisiae protein PubMed Scopus Google Scholar). The PTC7 gene and that of Snf2 leads to an increase in the of with is that the increase in the of in the and cells to be than the of previously that yeast Snf2 to on such as or M. the regulation of gene expression by in Saccharomyces PubMed Google Scholar). However, and the of such can Therefore, on and used as a control for the of the M. M. J. and of Full Text Full Text PDF PubMed Scopus Google of to previously as a role in CoQ6 in S. cerevisiae S. M. C. The phosphatase coenzyme biosynthesis by the in Full Text Full Text PDF PubMed Scopus Google Scholar). of the CoQ6 with 4-hydroxybenzoic as the and the role of is in is to CoQ6 biosynthesis via its of and of the of DMQ6, the of CoQ6 biosynthesis S. M. C. The phosphatase coenzyme biosynthesis by the in Full Text Full Text PDF PubMed Scopus Google S. M. C. coenzyme biosynthesis is regulated by a of J. PubMed Scopus Google Scholar). 4-hydroxybenzoic a for used to the of biosynthesis in yeast to The of Snf2 of CoQ6 and of there are significant changes in the of DMQ6, as well as 3-hexaprenyl-4-hydroxybenzoic an CoQ6 and with the of CoQ6 a of the yeast of to a significant increase in the of of to the by a target of S. M. C. The phosphatase coenzyme biosynthesis by the in Full Text Full Text PDF PubMed Scopus Google Scholar). we that the of and are in than in the yeast that the of Snf2 not CoQ6 by from that it the than a streamlined of of the to the of is by the that yeast of to yeast a than we the that the CoQ6 a of the in to of CoQ6 biosynthesis in and yeast at of of and of CoQ6 between and of in that there is an of in the yeast the of CoQ6 within of We of Snf2 as the and are with the role of Snf2 in decrease with in of in a that well with of Snf2 decrease with a increase in the splicing of PTC7 and splicing of the PTC7 transcript in yeast than in as Snf2 is from the splicing of the PTC7 transcript the of splicing in the the of CoQ6 a to the between and of with significant down-regulation in the of Snf2 protein is The decrease in the of Snf2 protein is in the increase in splicing of PTC7 transcript in the is to that the PTC7 transcript is spliced in the than in as the of Snf2 in the yeast splicing to a with the and and there is a increase in the CoQ6 and its biosynthesis in the yeast within of as with CoQ6 of the the and the increase in CoQ6 biosynthesis in the at by which the significant down-regulation in the of Snf2 protein is The and of and in the of and yeast and the of to CoQ6 increases by the of to in a with the decrease in Snf2 and increase in PTC7 splicing in with and In as the of Snf2 in yeast the of to CoQ6 the of to CoQ6 in yeast and The role of in the of CoQ6 in the of Snf2 can be from the that the from to CoQ6 is in the of the of not between and yeast the and However, the cells of and as well as of to and with the that of Snf2 the of by the of these to the that down-regulation of ribosomal protein genes of genes with splice the increase in PTC7 splicing is a of down-regulation used to target of transcription in a ribosomal protein gene expression via and the transcription Full Text Full Text PDF PubMed Scopus Google previously that mitochondrial in and in response to by J. J. M. in a of mitochondrial PubMed Scopus Google Scholar). In to a significant increase in the splicing of the PTC7 transcript and previously the in the of protein is than the in the of spliced to nonspliced transcript the of Snf2 that down-regulation changes the splicing of the PTC7 there are of gene regulation that control the of the and protein these mechanisms are these are with a down-regulation of expression spliceosomes from these abundant to otherwise poorly spliced such as PTC7 S. M. between for the splicing regulation of Cell. Full Text Full Text PDF PubMed Scopus Google S. S. A.R. T.L. The chromatin complex splicing of in Saccharomyces PubMed Scopus Google Scholar). In of the role of Snf2 in expression, changes in Snf2 of splicing in response to the The of the two of and and In the contains a encoded for by the PTC7 which is of the nuclear the of this is not to influence the of the of the its phosphatase the of isoform on CoQ6 we CoQ6 in cells of or there is significant in CoQ6 in the mitochondrial to Full Text Full Text PDF PubMed Scopus Google C. C. CoQ6 biosynthesis is for and in Cell. PubMed Scopus Google Scholar). However, expression of leads to an increase in CoQ6 expression of leads to a decrease in CoQ6 The RNA from are there are significant changes in the protein of Snf2 or the target of in these of these within the and from the PTC7 protein of the to be to the other and due to a that increases expression or of The and of CoQ6 in a with the yeast or and CoQ6 biosynthesis are in than in the and in to be that the two of and on CoQ6 biosynthesis In the of of as with the the of on CoQ6 biosynthesis is with the mechanisms of action it is that a on CoQ6 biosynthesis and in to the of this we the transcript of genes components of the CoQ6 complex and there is in the expression of the complex of Snf2 and or with the expression of or However, expression of is with down-regulation of of the and Although the by which these components are is it is that previously to the nuclear C. splicing of cerevisiae protein PubMed Scopus Google at a role for this isoform in expression of the RNAs the mechanisms by which may affect of the are via action on or via on gene is important to that to the of nuclear of, or a nuclear role in S. cerevisiae. Interestingly, yeast to or the to on a as not is with that of CoQ6 are for on a that CoQ6 are due to the of and in the under conditions of S. M. C. The phosphatase coenzyme biosynthesis by the in Full Text Full Text PDF PubMed Scopus Google M. S. of of protein phosphatase genes on in Saccharomyces PubMed Scopus Google Scholar). is that the of the or between the components of the or and CoQ6 not between isoform and is there are significant changes in the of as well as to and is of DMQ6, and HHB, with of these in and the between are and is with that the of Snf2 to the state of the in other in to its role in regulation of the a role for Snf2 in and in the transition from a fermentative of to a respiratory in S. as by the in conditions of high nutrient transcription of RPGs spliceosomes from with splice sites, such as a of the protein are at and on CoQ6 that CoQ6 is at a relatively the in the are the of Snf2 decrease spliceosomes to on leads to splicing of PTC7 and a in the of the two protein which leads to an increase in CoQ6 it that chromatin and chromatin factors influence splicing outcomes in the functional of such regulation under conditions a We previously that down-regulation of the of the SWI/SNF in response to nutrient leads to a in splicing outcomes due to down-regulation of RPGs and redistribution of spliceosomes S. M. between for the splicing regulation of Cell. Full Text Full Text PDF PubMed Scopus Google S. S. A.R. T.L. The chromatin complex splicing of in Saccharomyces PubMed Scopus Google Scholar). We that changes in splicing of PTC7 yeast with the conditions in the in the of a in the of two of the protein that and on CoQ6 biosynthesis. in the of the is with an increase in CoQ6 in the for the transition from a fermentative to a respiratory of metabolism. evidence the of PTC7 in CoQ6 biosynthesis. is for the of to its which is to the of the CoQ6 to the that the CoQ6 as by of from S. M. C. The phosphatase coenzyme biosynthesis by the in Full Text Full Text PDF PubMed Scopus Google Scholar). respiratory the of PTC7 not to a in CoQ6 as in lipid of cells mitochondrial to Full Text Full Text PDF PubMed Scopus Google The to this with the cells to the that the two in the under cells with the to and can the of We that expression of a significant on CoQ6 biosynthesis and the of from in the to the down-regulation of the and with we down-regulation of all of the components of the CoQ6 complex expression of The by which RNA expression is as and to the are PTC7 is not the of a gene in S. cerevisiae functional from the nonspliced pre-mRNA as well as the spliced C. splicing of cerevisiae protein PubMed Scopus Google Scholar). We translation of pre-mRNA leading to a functional in this translation from within the intron J. T.L. regulation of expression and is for in response to Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). the of the intron is the intron is in the of not it that of the protein are and is in the of S. a genome S. which the of of its two of the PTC7 Interestingly, the two PTC7 genes in a gene that encodes a mitochondrial from a spliced transcript of a within its and a gene a to to the nuclear from an nonspliced transcript of C. splicing and functional in PubMed Scopus Google Scholar). that protein from the PTC7 transcript in S. cerevisiae are to with of or C. splicing of cerevisiae protein PubMed Scopus Google Scholar). a role for in is that nuclear for numerous The of to in the gene regulation at to be for the of cells to and to nutrient in the regulation of gene Biochem. Full Text Full Text PDF PubMed Scopus Google Scholar). mitochondrial such as and components of the to significant nuclear in the regulation of gene expression of 27: PubMed Scopus Google of in cells with PubMed Scopus Google M. a mitochondrial and a of the PubMed Scopus Google of a protein of and mitochondrial ribosomal protein in mitochondrial translation in 2015; PubMed Scopus Google in response to PubMed Scopus Google Scholar). In of these of these to be to nuclear on reactions in Full Text PDF PubMed Google Scholar). with the of an isoform of in the nuclear the that a phosphatase is to regulation of components of the CoQ6 and the C. and of which is a target for in S. cerevisiae S. M. C. The phosphatase coenzyme biosynthesis by the in Full Text Full Text PDF PubMed Scopus Google to to the and are to of biosynthesis G.B. nuclear role for the respiratory in mitochondrial and 2015; PubMed Scopus Google Scholar). to with chromatin in cells G.B. nuclear role for the respiratory in mitochondrial and 2015; PubMed Scopus Google this to a C. S. the of the protein PubMed Scopus Google Scholar). nuclear of in S. cerevisiae not we a role in nuclear gene regulation for via phosphatase on or other nuclear and other mitochondrial is that a other than that expression of the In a with high numerous in a mitochondrial to Full Text Full Text PDF PubMed Scopus Google Scholar). this not between the of the two In expression of PTC7 not for a number of nuclear the of mitochondrial of PTC7 is completely by expression of mitochondrial to Full Text Full Text PDF PubMed Scopus Google Scholar). it is that the mitochondrial role for is in by and two other protein to the mitochondrial and a with the of S. M. C. The phosphatase coenzyme biosynthesis by the in Full Text Full Text PDF PubMed Scopus Google M. S. of of protein phosphatase genes on in Saccharomyces PubMed Scopus Google Scholar). Interestingly, the of Snf2 on CoQ6 biosynthesis not the expression of We that this is partially due to the Snf2 the of the CoQ6 as by the of of Snf2 on the expression of the components of the and it is the Snf2 may other of CoQ6 We are these the of Snf2 of yeast Snf2 a on such as or M. the regulation of gene expression by in Saccharomyces PubMed Google However, Snf2 protein is by in or as the not leads to that Snf2 protein is in response to it is for the transition from a fermentative state to that is respiratory in The of the for Snf2 in this transition are the of However, it is at least in part due to its role in the of genes transcription previously to occurs the or the yeast to a M. the regulation of gene expression by in Saccharomyces PubMed Google Scholar). We that the gene expression for to the nutrient the for Snf2 is and in the down-regulation of Snf2 splicing of for Snf2 to a report the role of Snf2 in at genes in it is to splicing of S. S. A.R. T.L. The chromatin complex splicing of in Saccharomyces PubMed Scopus Google Scholar). a by which SWI/SNF as a in the transition in S. cerevisiae. We of of a on the process of CoQ6 via of these mechanisms to be as well as in the gene regulation response to other conditions that yeast The yeast used in this are in are from the in yeast at Snf2 and with a expression or The by on the in with of a from C. splicing of cerevisiae protein PubMed Scopus Google Scholar). or and used for this are in The to on for than that it to from for all with the to a with to these We that this to the and of the and of yeast S. S. A.R. T.L. The chromatin complex splicing of in Saccharomyces PubMed Scopus Google S. S. A.R. T.L. The chromatin complex splicing of in Saccharomyces PubMed Scopus Google S. is a for the gene Full Text PDF PubMed Google C. of the S. cerevisiae genome by gene and PubMed Scopus Google in a The in this previously S. S. A.R. T.L. The chromatin complex splicing of in Saccharomyces PubMed Scopus Google Scholar). RNA the and ribosomal RNA sequence to and spliced from the M. yeast intron at PubMed Scopus Google in a J. C. S. M. PubMed Scopus Google Scholar). the for for at a for gene by the number of that within the by the in within ICGs as or within an as by the Introns with small RNAs within the intron in this are that with a that to an and partially within an and partially within an intron with and by spliced by the number of that to the spliced this is for this is the of the intron the for intron as spliced by the of the spliced and in splicing as and expression in are under number and previously S. S. A.R. T.L. The chromatin complex splicing of in Saccharomyces PubMed Scopus Google Scholar). RNA from a of to in of RNA from used to a PTC7 splicing the used for of on a and in a with of and a in for for the RNA RNA from expression by as a expression of with by of gene expression quantitative and the PubMed Scopus Google Scholar). from with and with The with protein by The to and with the at the at a in at a in at a in or previously of coenzyme in 2007; PubMed Scopus Google at a in with as by the in of in a and to an of in of the The as to an of and subsequently with at or control cells by at for from for lipid or for RNA and protein at of as C. J.A. of a coenzyme protein in the in Saccharomyces 2015; Full Text Full Text PDF PubMed Scopus Google with and and as the by and to to all with to to a and and and of lipid with a and a The of a with as and a with as The of from to the from to and conditions to conditions The as well as the for of the and in of and to an of in in which of and the The of the are and is a to protein and S. The for protein and 2015; PubMed Scopus Google Scholar). is used to the sequence of a protein with a that the protein of with other of with In to the nonspliced of the protein which is composed of and its intron The and of at with of the spliced isoform of which is and in the of from the removal of the intron and the of the mitochondrial sequence of S. C. J. C. of the mitochondrial a critical for protein Full Text Full Text PDF PubMed Scopus Google the The and contains of at with of M. and S. to this the of the the and the and M. C. M. in M. in G. and S. in with G. in the to PTC7 splicing in the in S. the and splicing M. C. and and the of the C. C. and J. all to the and on and the of this We for and in the and in the of the spliced and nonspliced of We the for the of the We the of for the spliced and nonspliced

Abstract The dynamic translocation of transcription factors (TFs) in and out of the nucleus is thought to encode information, such as the identity of a stimulus. A corollary is the idea that gene promoters can decode different dynamic TF translocation patterns. Testing this TF encoding/promoter decoding hypothesis requires tools that allow direct control of TF dynamics without the pleiotropic effects associated with general perturbations. In this work, we present CLASP (Controllable Light Activated Shuttling and Plasma membrane sequestration), a tool that enables precise, modular, and reversible control of TF localization using a combination of two optimized LOV2 optogenetic constructs. The first sequesters the cargo in the dark at the plasma membrane and releases it upon exposure to blue light, while light exposure of the second reveals a nuclear localization sequence that shuttles the released cargo to the nucleus. CLASP achieves minute-level resolution, reversible translocation of many TF cargos, large dynamic range, and tunable target gene expression. Using CLASP, we investigate the relationship between Crz1, a naturally pulsatile TF, and its cognate promoters. We establish that some Crz1 target genes respond more efficiently to pulsatile TF inputs than to continuous inputs, while others exhibit the opposite behavior. We show using computational modeling that efficient gene expression in response to short pulsing requires fast promoter activation and slow inactivation and that the opposite phenotype can ensue from a multi-stage promoter activation, where a transition in the first stage is thresholded. These data directly demonstrate differential interpretation of TF pulsing dynamics by different genes, and provide plausible models that can achieve these phenotypes.