scholarly journals Repositioning the Sm-binding site in S. cerevisiae telomerase RNA reveals RNP organizational flexibility and Sm-directed 3′-end formation

2017 ◽  
Author(s):  
Evan P. Hass ◽  
David C. Zappulla
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Strong Evidence ◽  
Binding Sites ◽  
Telomerase Rna ◽  
Telomeric Dna ◽  
Protein Subunits ◽  
Yeast Telomerase ◽  
Rna Biogenesis

ABSTRACTTelomerase RNA contains a template for synthesizing telomeric DNA by reverse transcription and has been proposed to act as a flexible scaffold for holoenzyme protein subunits in the RNP. In Saccharomyces cerevisiae, the telomerase subunits Est1 and Ku bind to the telomerase RNA, TLC1, and it has been shown that these proteins still function when their binding sites are repositioned within the RNA. TLC1 is also bound by the Sm7 protein complex, which is required for stabilization of the predominant, non-polyadenylated (poly(A)–) TLC1 isoform. Here, we first show that Sm7 can perform this function even when its binding site is repositioned via circular permutation to several different positions within TLC1, further supporting the conclusion that the telomerase holoenzyme is organizationally flexible. Next, we tested the hypothesis that the location of the Sm7-binding site relative to the 3′ end is contrastingly important. When we moved the Sm site to locations 5′ of its native position, we observed that this stabilized shorter forms of poly(A)– TLC1 in a manner precisely corresponding to how far upstream the Sm site was moved. This provides strong evidence that the location of Sm7 binding to TLC1 controls where the mature poly(A)– 3′ end is formed. In summary, our results show that Sm7 and the 3′ end of yeast telomerase RNA comprise an organizationally flexible module within the telomerase RNP and provide insights into the mechanistic role of Sm7 in telomerase RNA biogenesis.

scholarly journals Repositioning the Sm-Binding Site in Saccharomyces cerevisiae Telomerase RNA Reveals RNP Organizational Flexibility and Sm-Directed 3′-End Formation

2020 ◽  
Vol 6 (1) ◽  
pp. 9 ◽  
Author(s):  
Evan P. Hass ◽  
David C. Zappulla
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Telomerase Rna ◽  
Telomeric Dna ◽  
Protein Subunits ◽  
Yeast Telomerase ◽  
Rna Biogenesis ◽  
Native Site ◽  
Processing Mechanism

Telomerase RNA contains a template for synthesizing telomeric DNA and has been proposed to act as a flexible scaffold for holoenzyme protein subunits in the RNP. In Saccharomyces cerevisiae, the telomerase RNA, TLC1, is bound by the Sm7 protein complex, which is required for stabilization of the predominant, non-polyadenylated (poly(A)–) TLC1 isoform. However, it remains unclear (1) whether Sm7 retains this function when its binding site is repositioned within TLC1, as has been shown for other TLC1-binding telomerase subunits, and (2) how Sm7 stabilizes poly(A)– TLC1. Here, we first show that Sm7 can stabilize poly(A)– TLC1 even when its binding site is repositioned via circular permutation to several different positions within TLC1, further supporting the conclusion that the telomerase holoenzyme is organizationally flexible. Next, we show that when an Sm site is inserted 5′ of its native position and the native site is mutated, Sm7 stabilizes shorter forms of poly(A)– TLC1 in a manner corresponding to how far upstream the new site was inserted, providing strong evidence that Sm7 binding to TLC1 controls where the mature poly(A)– 3′ is formed by directing a 3′-to-5′ processing mechanism. In summary, our results show that Sm7 and the 3′ end of yeast telomerase RNA comprise an organizationally flexible module within the telomerase RNP and provide insights into the mechanistic role of Sm7 in telomerase RNA biogenesis.

scholarly journals Essential Regions of Saccharomyces cerevisiae Telomerase RNA: Separate Elements for Est1p and Est2p Interaction

2002 ◽  
Vol 22 (7) ◽  
pp. 2366-2374 ◽  
Author(s):  
April J. Livengood ◽  
Arthur J. Zaug ◽  
Thomas R. Cech
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nucleotide Sequence ◽  
Dna Synthesis ◽  
Active Site ◽  
Telomerase Rna ◽  
Telomeric Dna ◽  
Protein Subunits ◽  
Essential Region ◽  
Selection For

ABSTRACT The Saccharomyces cerevisiae telomerase RNA subunit is encoded by the TLC1 gene. A selection for viable alleles of TLC1 RNA from a large library of random deletion alleles revealed that less than half (∼0.5 kb of the ∼1.3-kb RNA) is required for telomerase function in vivo. The main essential region (430 nucleotides), which contains the template for telomeric DNA synthesis, was required for coimmunoprecipitation with Est1p and Est2p. Furthermore, the subregion required for interaction with Est1p, the telomerase recruitment subunit, differed from those required for interaction with Est2p, the reverse transcriptase subunit. Two regions of the RNA distant from the template in the nucleotide sequence were required for Est2p binding, but the template itself was not. Having the RNA secured to the protein away from the template is proposed to facilitate the translocation of the RNA template through the active site. More generally, our results support a role for the telomerase RNA serving as a scaffold for binding key protein subunits.

scholarly journals Yeast Telomerase RNA Flexibly Scaffolds Protein Subunits: Results and Repercussions

Molecules ◽  
2020 ◽  
Vol 25 (12) ◽  
pp. 2750 ◽  
Author(s):  
David C. Zappulla
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Noncoding Rna ◽  
Noncoding Rnas ◽  
Telomerase Rna ◽  
Protein Subunits ◽  
Rna Molecules ◽  
Yeast Telomerase ◽  
Opportune Time ◽  
Rnp Complex ◽  
Functional Features

It is said that “hindsight is 20-20,” so, given the current year, it is an opportune time to review and learn from experiences studying long noncoding RNAs. Investigation of the Saccharomyces cerevisiae telomerase RNA, TLC1, has unveiled striking flexibility in terms of both structural and functional features. Results support the “flexible scaffold” hypothesis for this 1157-nt telomerase RNA. This model describes TLC1 acting as a tether for holoenzyme protein subunits, and it also may apply to a plethora of RNAs beyond telomerase, such as types of lncRNAs. In this short perspective review, I summarize findings from studying the large yeast telomerase ribonucleoprotein (RNP) complex in the hope that this hindsight will sharpen foresight as so many of us seek to mechanistically understand noncoding RNA molecules from vast transcriptomes.

scholarly journals The Est1 Subunit of Yeast Telomerase Binds the Tlc1 Telomerase RNA

2000 ◽  
Vol 20 (6) ◽  
pp. 1947-1955 ◽  
Author(s):  
Jianlong Zhou ◽  
Kyoko Hidaka ◽  
Bruce Futcher
Keyword(s):  
Point Mutations ◽  
Telomerase Activity ◽  
Rna Recognition Motif ◽  
Telomerase Rna ◽  
Telomeric Dna ◽  
Reading Frame ◽  
Yeast Telomerase ◽  
Exact Function ◽  
Telomere Loss

ABSTRACT Est1 is a component of yeast telomerase, and est1mutants have senescence and telomere loss phenotypes. The exact function of Est1 is not known, and it is not homologous to components of other telomerases. We previously showed that Est1 protein coimmunoprecipitates with Tlc1 (the telomerase RNA) as well as with telomerase activity. Est1 has homology to Ebs1, an uncharacterized yeast open reading frame product, including homology to a putative RNA recognition motif (RRM) of Ebs1. Deletion of EBS1 results in short telomeres. We created point mutations in a putative RRM of Est1. One mutant was unable to complement either the senescence or the telomere loss phenotype of est1 mutants. Furthermore, the mutant protein no longer coprecipitated with the Tlc1 telomerase RNA. Mutants defective in the binding of Tlc1 RNA were nevertheless capable of binding single-stranded TG-rich DNA. Our data suggest that an important role of Est1 in the telomerase complex is to bind to the Tlc1 telomerase RNA via an RRM. Since Est1 can also bind telomeric DNA, Est1 may tether telomerase to the telomere.

scholarly journals Two Compound Replication Origins in Saccharomyces cerevisiae Contain Redundant Origin Recognition Complex Binding Sites

2001 ◽  
Vol 21 (8) ◽  
pp. 2790-2801 ◽  
Author(s):  
James F. Theis ◽  
Carol S. Newlon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Binding Site ◽  
Binding Sites ◽  
Origin Recognition Complex ◽  
Replication Origins ◽  
Discrete Site ◽  
Origin Function ◽  
Single Binding Site ◽  
Origin Recognition

ABSTRACT While many of the proteins involved in the initiation of DNA replication are conserved between yeasts and metazoans, the structure of the replication origins themselves has appeared to be different. As typified by ARS1, replication origins inSaccharomyces cerevisiae are <150 bp long and have a simple modular structure, consisting of a single binding site for the origin recognition complex, the replication initiator protein, and one or more accessory sequences. DNA replication initiates from a discrete site. While the important sequences are currently less well defined, metazoan origins appear to be different. These origins are large and appear to be composed of multiple, redundant elements, and replication initiates throughout zones as large as 55 kb. In this report, we characterize two S. cerevisiae replication origins, ARS101 and ARS310, which differ from the paradigm. These origins contain multiple, redundant binding sites for the origin recognition complex. Each binding site must be altered to abolish origin function, while the alteration of a single binding site is sufficient to inactivate ARS1. This redundant structure may be similar to that seen in metazoan origins.

Identification of SAS4 and SAS5, Two Genes That Regulate Silencing in Saccharomyces cerevisiae

Genetics ◽  
1999 ◽  
Vol 153 (1) ◽  
pp. 13-23 ◽  
Author(s):  
Eugenia Y Xu ◽  
Susan Kim ◽  
Kirstin Replogle ◽  
Jasper Rine ◽  
David H Rivier
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acute Myeloid Leukemia ◽  
Binding Site ◽  
Myeloid Leukemia ◽  
Null Alleles ◽  
Human Genes ◽  
Complementation Groups ◽  
A Site ◽  
Acute Myeloid

Abstract In Saccharomyces cerevisiae, chromatin-mediated silencing inactivates transcription of the genes at the HML and HMR cryptic mating-type loci and genes near telomeres. Mutations in the Rap1p and Abf1p binding sites of the HMR-E silencer (HMRa-e**) result in a loss of silencing at HMR. We characterized a collection of 15 mutations that restore the α-mating phenotype to MATα HMRa-e** strains. These mutations defined three complementation groups, two new groups and one group that corresponded to the previously identified SAS2 gene. We cloned the genes that complemented members of the new groups and identified two previously uncharacterized genes, which we named SAS4 and SAS5. Neither SAS4 nor SAS5 was required for viability. Null alleles of SAS4 and SAS5 restored SIR4-dependent silencing at HMR, establishing that each is a regulator of silencing. Null alleles of SAS4 and SAS5 bypassed the role of the Abf1p binding site of the HMR-E silencer but not the role of the ACS or Rap1p binding site. Previous analysis indicated that SAS2 is homologous to a human gene that is a site of recurring translocations involved in acute myeloid leukemia. Similarly, SAS5 is a member of a gene family that included two human genes that are the sites of recurring translocations involved in acute myeloid leukemia.

A synthetic silencer mediates SIR-dependent functions in Saccharomyces cerevisiae

1991 ◽  
Vol 11 (11) ◽  
pp. 5648-5659
Author(s):  
F J McNally ◽  
J Rine
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Binding Sites ◽  
Consensus Sequence ◽  
Protein A ◽  
Replication Initiation ◽  
Yeast Saccharomyces Cerevisiae ◽  
Autonomously Replicating Sequence ◽  
Functional Components

Copies of the mating-type genes are present at three loci on chromosome III of the yeast Saccharomyces cerevisiae. The genes at the MAT locus are transcribed, whereas the identical genes at the silent loci, HML and HMR, are not transcribed. Several genes, including the four SIR genes, and two sites, HMR-E and HMR-I, are required for repression of transcription at the HMR locus. Three elements have been implicated in the function of the HMR-E silencer: a binding site for the RAP1 protein, a binding site for the ABF1 protein, and an 11-bp consensus sequence common to nearly all autonomously replicating sequence (ARS) elements (putative origins of DNA replication). RAP1 and ABF1 binding sites of different sequence than those found at HMR-E were joined with an 11-bp ARS consensus sequence to form a synthetic silencer. The synthetic silencer was able to repress transcription of the HMRa1 gene, confirming that binding sites for RAP1 and ABF1 and the 11-bp ARS consensus sequence were the functional components of the silencer in vivo. Mutations in the ABF1 binding site or in the ARS consensus sequence of the synthetic silencer caused nearly complete derepression of transcription at HMR. The ARS consensus sequence mutation also eliminated the ARS activity of the synthetic silencer. These data suggested that replication initiation at the HMR-E silencer was required for establishment of the repressed state at the HMR locus.

scholarly journals The C Terminus of the Major Yeast Telomere Binding Protein Rap1p Enhances Telomere Formation

1998 ◽  
Vol 18 (3) ◽  
pp. 1284-1295 ◽  
Author(s):  
Alo Ray ◽  
Kurt W. Runge
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Reverse Transcriptase ◽  
Binding Site ◽  
Binding Protein ◽  
Telomerase Activity ◽  
Telomerase Rna ◽  
Constant Length ◽  
C Terminus ◽  
Telomere Binding Protein ◽  
Yeast Telomere

ABSTRACT The telomeres of most organisms consist of short repeated sequences that can be elongated by telomerase, a reverse transcriptase complex that contains its own RNA template for the synthesis of telomere repeats. In Saccharomyces cerevisiae, the RAP1gene encodes the major telomere binding protein Rap1p. Here we use a quantitative telomere formation assay to demonstrate that Rap1p C termini can enhance telomere formation more than 30-fold when they are located at internal sites. This stimulation is distinct from protection from degradation. Enhancement of formation required the gene for telomerase RNA but not Sir1p, Sir2p, Sir3p, Sir4p, Tel1p, or the Rif1p binding site in the Rap1p C terminus. Our data suggest that Rap1p C termini enhance telomere formation by attracting or increasing the activity of telomerase near telomeres. Earlier work suggests that Rap1p molecules at the chromosome terminus inhibit the elongation of long telomeres by blocking the access of telomerase. Our results suggest a model where a balance between internal Rap1p increasing telomerase activity and Rap1p at the termini of long telomeres controlling telomerase access maintains telomeres at a constant length.

scholarly journals Characterization of the DNA-binding activity of GCR1: in vivo evidence for two GCR1-binding sites in the upstream activating sequence of TPI of Saccharomyces cerevisiae.

1992 ◽  
Vol 12 (6) ◽  
pp. 2690-2700 ◽  
Author(s):  
M A Huie ◽  
E W Scott ◽  
C M Drazinic ◽  
M C Lopez ◽  
I K Hornstra ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fusion Protein ◽  
Binding Site ◽  
Gene Function ◽  
Binding Sites ◽  
Sequence Motif ◽  
Wild Type ◽  
Upstream Activating Sequence ◽  
Sequence Elements

GCR1 gene function is required for high-level glycolytic gene expression in Saccharomyces cerevisiae. Recently, we suggested that the CTTCC sequence motif found in front of many genes encoding glycolytic enzymes lay at the core of the GCR1-binding site. Here we mapped the DNA-binding domain of GCR1 to the carboxy-terminal 154 amino acids of the polypeptide. DNase I protection studies showed that a hybrid MBP-GCR1 fusion protein protected a region of the upstream activating sequence of TPI (UASTPI), which harbored the CTTCC sequence motif, and suggested that the fusion protein might also interact with a region of the UAS that contained the related sequence CATCC. A series of in vivo G methylation protection experiments of the native TPI promoter were carried out with wild-type and gcr1 deletion mutant strains. The G doublets that correspond to the C doublets in each site were protected in the wild-type strain but not in the gcr1 mutant strain. These data demonstrate that the UAS of TPI contains two GCR1-binding sites which are occupied in vivo. Furthermore, adjacent RAP1/GRF1/TUF- and REB1/GRF2/QBP/Y-binding sites in UASTPI were occupied in the backgrounds of both strains. In addition, DNA band-shift assays were used to show that the MBP-GCR1 fusion protein was able to form nucleoprotein complexes with oligonucleotides that contained CTTCC sequence elements found in front of other glycolytic genes, namely, PGK, ENO1, PYK, and ADH1, all of which are dependent on GCR1 gene function for full expression. However, we were unable to detect specific interactions with CTTCC sequence elements found in front of the translational component genes TEF1, TEF2, and CRY1. Taken together, these experiments have allowed us to propose a consensus GCR1-binding site which is 5'-(T/A)N(T/C)N(G/A)NC(T/A)TCC(T/A)N(T/A)(T/A)(T/G)-3'.

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