Christopher J. Webb

Identification and characterization of the Schizosaccharomyces pombe TER1 telomerase RNA

Although the catalytic subunit of the Schizosaccharomyces pombe telomerase holoenzyme was identified over ten years ago, the unusual heterogeneity of its telomeric DNA made it difficult to identify its RNA component. We used a new two-step immunoprecipitation and reverse transcription–PCR technique to identify the S. pombe telomerase RNA, which we call TER1. TER1 RNA was 1,213 nucleotides long, similar in size to the Saccharomyces cerevisiae telomerase RNA, TLC1. TER1 RNA associated in vivo with the two known subunits of the S. pombe telomerase holoenzyme, Est1p and Trt1p, and neither association was dependent on the other holoenzyme component. We present a model to explain how telomerase introduces heterogeneity into S. pombe telomeres. The technique used here to identify TER1 should be generally applicable to other model organisms.

Evidence for Splice Site Pairing via Intron Definition in Schizosaccharomyces pombe

ABSTRACT Schizosaccharomyces pombe pre-mRNAs are generally multi-intronic and share certain features with pre-mRNAs fromDrosophila melanogaster, in which initial splice site pairing can occur via either exon or intron definition. Here, we present three lines of evidence suggesting that, despite these similarities, fission yeast splicing is most likely restricted to intron definition. First, mutating either or both splice sites flanking an internal exon in the S. pombe cdc2 gene produced almost exclusively intron retention, in contrast to the exon skipping observed in vertebrates. Second, we were unable to induce skipping of the internal microexon in fission yeast cgs2, whereas the default splicing pathway excludes extremely small exons in mammals. Because nearly quantitative removal of the downstream intron incgs2 could be achieved by expanding the microexon, we propose that its retention is due to steric occlusion. Third, several cryptic 5′ junctions in the second intron of fission yeastcdc2 are located within the intron, in contrast to their generally exonic locations in metazoa. The effects of expanding and contracting this intron are as predicted by intron definition; in fact, even highly deviant 5′ junctions can compete effectively with the standard 5′ splice site if they are closer to the 3′ splicing signals. Taken together, our data suggest that pairing of splice sites inS. pombe most likely occurs exclusively across introns in a manner that favors excision of the smallest segment possible.

DNA replication through hard-to-replicate sites, including both highly transcribed RNA Pol II and Pol III genes, requires the S. pombe Pfh1 helicase

Replication forks encounter impediments as they move through the genome, including natural barriers due to stable protein complexes and highly transcribed genes. Unlike lesions generated by exogenous damage, natural barriers are encountered in every S phase. Like humans, Schizosaccharomyces pombe encodes a single Pif1 family DNA helicase, Pfh1. Here, we show that Pfh1 is required for efficient fork movement in the ribosomal DNA, the mating type locus, tRNA, 5S ribosomal RNA genes, and genes that are highly transcribed by RNA polymerase II. In addition, converged replication forks accumulated at all of these sites in the absence of Pfh1. The effects of Pfh1 on DNA replication are likely direct, as it had high binding to sites whose replication was impaired in its absence. Replication in the absence of Pfh1 resulted in DNA damage specifically at those sites that bound high levels of Pfh1 in wild-type cells and whose replication was slowed in its absence. Cells depleted of Pfh1 were inviable if they also lacked the human TIMELESS homolog Swi1, a replisome component that stabilizes stalled forks. Thus, Pfh1 promotes DNA replication and separation of converged replication forks and suppresses DNA damage at hard-to-replicate sites.

Schizosaccharomyces pombe Ccq1 and TER1 bind the 14-3-3-like domain of Est1, which promotes and stabilizes telomerase–telomere association

The telomerase protein Est1 exists in multiple organisms, including Schizosaccharomyces pombe, humans, and Saccharomyces cerevisiae, but its function has only been closely examined in S. cerevisiae, where it is a recruiter/activator of telomerase. Here, we demonstrate that S. pombe Est1 was required for the telomere association of the telomerase holoenzyme, suggesting that it too has a recruitment role. Its association with telomeres was dependent on Trt1, the catalytic subunit, and Ccq1, a telomeric protein. Surprisingly, Est1 telomere binding was only partially dependent on TER1, the telomerase RNA, even though Est1 bound nucleotides 415-507 of TER1. A ter1-Δ415-507 strain had short telomeres and very low Est1 and Trt1 telomere association in late S phase but did not senesce. An unbiased search for mutations that reduced Est1-TER1 interaction identified mutations only in the Est1 14-3-3-like domain, a phosphoserine-binding motif, the first example of a 14-3-3-like domain with RNA-binding activity. These mutations also reduced Est1-Ccq1 binding. One such mutant prevented Est1 telomere association and caused telomere loss and slow senescence, similar to ccq1Δ. We propose that the Est1-Ccq1 interaction is critical for telomerase recruitment, while the Est1-TER1 interaction acts downstream from Ccq1-mediated recruitment to stabilize the holoenzyme at the telomere.

The Splicing Factor U2AF Small Subunit Is Functionally Conserved between Fission Yeast and Humans

ABSTRACT The small subunit of U2AF, which functions in 3′ splice site recognition, is more highly conserved than its heterodimeric partner yet is less thoroughly investigated. Remarkably, we find that the small subunit of Schizosaccharomyces pombe U2AF (U2AFSM) can be replaced in vivo by its human counterpart, demonstrating that the conservation extends to function. Precursor mRNAs accumulate in S. pombe following U2AFSM depletion in a time frame consistent with a role in splicing. A comprehensive mutational analysis reveals that all three conserved domains are required for viability. Notably, however, a tryptophan in the pseudo-RNA recognition motif implicated in a key contact with the large subunit by crystallographic data is dispensable whereas amino acids implicated in RNA recognition are critical. Mutagenesis of the two zinc-binding domains demonstrates that they are neither equivalent nor redundant. Finally, two- and three-hybrid analyses indicate that mutations with effects on large-subunit interactions are rare whereas virtually all alleles tested diminished RNA binding by the heterodimer. In addition to demonstrating extraordinary conservation of U2AF small-subunit function, these results provide new insights into the roles of individual domains and residues.

Telomerase RNA stem terminus element affects template boundary element function, telomere sequence, and shelterin binding

Significance We demonstrate that the fission yeast telomerase RNA has a stem terminus element (STE) that it is essential for telomerase action in vivo and in vitro. Using a partial loss-of-function STE allele, we show that the STE is required for wild-type telomeric sequence. This is the first example, to our knowledge, of a sequence that is not part of the telomerase RNA core region that affects the sequence of telomeric DNA. Because mutating the STE has the same phenotypes as mutating the template boundary element (TBE), the STE promotes TBE function. The association of two sequence-specific telomere binding proteins is impaired in the STE mutant. Thus, the STE is critical to assemble the normal sequence and chromatin structure of fission yeast telomeres. The stem terminus element (STE), which was discovered 13 y ago in human telomerase RNA, is required for telomerase activity, yet its mode of action is unknown. We report that the Schizosaccharomyces pombe telomerase RNA, TER1 (telomerase RNA 1), also contains a STE, which is essential for telomere maintenance. Cells expressing a partial loss-of-function TER1 STE allele maintained short stable telomeres by a recombination-independent mechanism. Remarkably, the mutant telomere sequence was different from that of wild-type cells. Generation of the altered sequence is explained by reverse transcription into the template boundary element, demonstrating that the STE helps maintain template boundary element function. The altered telomeres bound less Pot1 (protection of telomeres 1) and Taz1 (telomere-associated in Schizosaccharomyces pombe 1) in vivo. Thus, the S. pombe STE, although distant from the template, ensures proper telomere sequence, which in turn promotes proper assembly of the shelterin complex.

Telomerase RNA is more than a DNA template

ABSTRACT The addition of telomeric DNA to chromosome ends is an essential cellular activity that compensates for the loss of genomic DNA that is due to the inability of the conventional DNA replication apparatus to duplicate the entire chromosome. The telomerase reverse transcriptase and its associated RNA bind to the very end of the telomere via a sequence in the RNA and specific protein-protein interactions. Telomerase RNA also provides the template for addition of new telomeric repeats by the reverse-transcriptase protein subunit. In addition to the template, there are 3 other conserved regions in telomerase RNA that are essential for normal telomerase activity. Here we briefly review the conserved core regions of telomerase RNA and then focus on a recent study in fission yeast that determined the function of another conserved region in telomerase RNA called the Stem Terminus Element (STE).1 The STE is distant from the templating core of telomerase in both the linear and RNA secondary structure, but, nonetheless, affects the fidelity of telomere sequence addition and, in turn, the ability of telomere binding proteins to bind and protect chromosome ends. We will discuss possible mechanisms of STE action and the suitability of the STE as an anti-cancer target.

Telomere les(i/s)ons from a telomerase RNA mutant

The “end replication problem,” the inability of conventional DNA polymerases to duplicate the very ends of linear DNA molecules, is usually solved by telomerase, a specialized reverse transcriptase. The telomerase holoenzyme consists of the catalytic subunit (TERT), a telomerase RNA (TER) and species-specific accessory factors. Although telomerase RNAs vary enormously in size and nucleotide sequence, 4 structures are highly conserved: (1) a telomere repeat templating sequence, which is single-stranded in the folded RNA; (2) a template boundary element (TBE) helix that is immediately adjacent to the template and prevents synthesis past the template; (3) a pseudoknot that affects template usage, and (4) the stem terminus element (STE), which was discovered in mammals in 2000 where it is essential for telomerase activity in vitro and in vivo yet its mode of action was previously unknown. Except for the STE, the 3 other elements are in the catalytic core region of the RNA. This review summarizes a recent paper that takes advantage of the conservation of STE sequence and structure in the fission yeast Schizosaccharomyces pombe (Fig. 1) to reveal the function of the STE. To understand how the STE works, we generated a panel of fission yeast STE mutants and determined their effects on telomere length. This characterization showed that, as in mammals, the fission yeast STE is essential for telomerase activity and telomere maintenance and identified a partial-loss-of-function allele, ter1-STEloop. In this mutant, the 4 tandem uridines in the STE tetraloop are changed to adenines (Fig. 1). Because telomeres are short but stable in ter1-STEloop cells, the mutant was ideal for analyzing STE function in vivo. Although non-canonical telomere repeats are seen in most organisms, fission yeast telomeric DNA is particularly heterogeneous, as reflected in its consensus sequence, 50-(G)0–6GGTTACAC-30 (the rare cytosine, discussed below, is underlined). Our ability to isolate the ter1-STEloop allele was probably facilitated by this heterogeneity, which arises from the inefficiency of the fission yeast TBE, which is less capable than TBEs in other organisms at preventing run-on reverse transcription. In wild type fission yeast, occasional TBE failure results in reverse transcription into the TBE, which introduces a rare cytosine at the end of the core telomere sequence in about 12% of repeats. The variable number of guanine residues is generated by “stuttering” that arises from the absence of base-pairing between the RNA template and the telomeric DNA end. Because incorporation of the rare cytosine changes the template RNA-telomeric DNA register, its addition results in reduced stuttering and hence fewer guanine-tracts (Reviewed in). Although we considered that the STE promotes holoenzyme formation, TERT association with TER1 was unaffected in ter1-STEloop cells. Likewise, the physical interaction between telomerase and the telomere was normal. However, as in humans, telomerase catalysis is compromised as extracts from ter1-STEloop cells had very low activity. We isolated and cloned telomeric DNA from mutant and wild type cells and determined their sequence. For cloning, we developed a novel method that exploited the rapid but reversible telomere shortening that occurs by growing fission yeast cells at higher temperatures. This approach ensured that we analyzed only the sequence templated by TER1-STEloop. Surprisingly, ter1-STEloop telomeric DNA does not have wild type sequence. Rather, the mutant telomeres have a much higher frequency of rare cytosines and reduced frequency of guanine tracts, 2 phenotypes first seen in cells with mutant TBEs (Fig. 1). The unusual sequence of ter1-STEloop telomeric DNA was recombination independent but telomerase dependent. Thus, the STE affects how the telomerase catalytic core templates nucleotide addition. Because the ter1-STEloop mutation affects the sequence of telomeric DNA in the same way as mutations that disrupt TBE function, we conclude that the STE enforces TBE activity. The TER1-STEloop templated change in telomeric sequence also affected telomeric chromatin. In fission yeast and mammals, telomeres are bound by the multi-subunit shelterin complex, which together with the telomeric DNA, protects telomeres and mediates all telomere functions. Using chromatin immunoprecipitaton, we analyzed telomere binding of