Protein Hit1, a novel box C/D snoRNP assembly factor,controls cellular concentration of the scaffolding protein Rsa1 by direct interaction

Biogenesis of eukaryotic box C/D small nucleolar ribonucleoprotein particles (C/D snoRNPs) involves conserved trans-acting factors, which are proposed to facilitate the assembly of the core proteins Snu13p/15.5K, Nop58p/NOP58, Nop56p/NOP56 and Nop1p/Fibrillarin on box C/D small nucleolar RNAs (C/D snoRNAs). In yeast, protein Rsa1 acts as a platform, interacting with both the RNA-binding core protein Snu13 and protein Pih1 of the Hsp82–R2TP chaperone complex. In this work, a proteomic approach coupled with functional and structural studies identifies protein Hit1 as a novel Rsa1p-interacting partner involved in C/D snoRNP assembly. Hit1p contributes to in vivo C/D snoRNA stability and pre-RNA maturation kinetics. It associates with U3 snoRNA precursors and influences its 3′-end processing. Remarkably, Hit1p is required to maintain steady-state levels of Rsa1p. This stabilizing activity is likely to be general across eukaryotic species, as the human protein ZNHIT3(TRIP3) showing sequence homology with Hit1p regulates the abundance of NUFIP1, the Rsa1p functional homolog. The nuclear magnetic resonance solution structure of the Rsa1p_317–352–Hit1p_70–164 complex reveals a novel mode of protein–protein association explaining the strong stability of the Rsa1p–Hit1p complex. Our biochemical data show that C/D snoRNAs and the core protein Nop58 can interact with the purified Snu13p–Rsa1p–Hit1p heterotrimer.     

Career guidance for stem cells

In vitro, assembly of box C/D small nucleolar ribonucleoproteins (snoRNPs) involves the sequential recruitment of core proteins to snoRNAs. In vivo, however, assembly factors are required (NUFIP, BCD1, and the HSP90–R2TP complex), and it is unknown whether a similar sequential scheme applies. In this paper, we describe systematic quantitative stable isotope labeling by amino acids in cell culture proteomic experiments and the crystal structure of the core protein Snu13p/15.5K bound to a fragment of the assembly factor Rsa1p/NUFIP. This revealed several unexpected features: (a) the existence of a protein-only pre-snoRNP complex containing five assembly factors and two core proteins, 15.5K and Nop58; (b) the characterization of ZNHIT3, which is present in the protein-only complex but gets released upon binding to C/D snoRNAs; (c) the dynamics of the R2TP complex, which appears to load/unload RuvBL AAA⁺ adenosine triphosphatase from pre-snoRNPs; and (d) a potential mechanism for preventing premature activation of snoRNP catalytic activity. These data provide a framework for understanding the assembly of box C/D snoRNPs.     

A dynamic scaffold of pre-snoRNP factors facilitates human box C/D snoRNP assembly

The box C/D small nucleolar RNPs (snoRNPs) are essential for the processing and modification of rRNA. The core box C/D proteins are restructured during human U3 box C/D snoRNP biogenesis; however, the molecular basis of this is unclear. Here we show that the U8 snoRNP is also restructured, suggesting that this may occur with all box C/D snoRNPs. We have characterized four novel human biogenesis factors (BCD1, NOP17, NUFIP, and TAF9) which, along with the ATPases TIP48 and TIP49, are likely to be involved in the formation of the pre-snoRNP. We have analyzed the in vitro protein-protein interactions between the assembly factors and core box C/D proteins. Surprisingly, this revealed few interactions between the individual core box C/D proteins. However, the novel biogenesis factors and TIP48 and TIP49 interacted with one or more of the core box C/D proteins, implying that they mediate the assembly of the pre-snoRNP. Consistent with this, we show that NUFIP bridges interactions between the core box C/D proteins in a partially reconstituted pre-snoRNP. Restructuring of the core complex probably reflects the conversion of the pre-snoRNP, where core protein-protein interactions are maintained by the bridging biogenesis factors, to the mature snoRNP.     

A common core RNP structure shared between the small nucleoar box C/D RNPs and the spliceosomal U4 snRNP

The box C/D snoRNAs function in directing 2'-O-methylation and/or as chaperones in the processing of ribosomal RNA. We show here that Snu13p (15.5 kD in human), a component of the U4/U6.U5 tri-snRNP, is also associated with the box C/D snoRNAs. Indeed, genetic depletion of Snu13p in yeast leads to a major defect in RNA metabolism. The box C/D motif can be folded into a stem-internal loop-stem structure, almost identical to the 15.5 kD binding site in the U4 snRNA. Consistent with this, the box C/D motif binds Snu13p/ 15.5 kD in vitro. The similarities in structure and function observed between the U4 snRNP (chaperone for U6) and the box C/D snoRNPs raises the interesting possibility that these particles may have evolved from a common ancestral RNP.     

NUFIP and the HSP90/R2TP chaperone bind the SMN complex and facilitate assembly of U4-specific proteins

The Sm proteins are loaded on snRNAs by the SMN complex, but how snRNP-specific proteins are assembled remains poorly characterized. U4 snRNP and box C/D snoRNPs have structural similarities. They both contain the 15.5K and proteins with NOP domains (PRP31 for U4, NOP56/58 for snoRNPs). Biogenesis of box C/D snoRNPs involves NUFIP and the HSP90/R2TP chaperone system and here, we explore the function of this machinery in U4 RNP assembly. We show that yeast Prp31 interacts with several components of the NUFIP/R2TP machinery, and that these interactions are separable from each other. In human cells, PRP31 mutants that fail to stably associate with U4 snRNA still interact with components of the NUFIP/R2TP system, indicating that these interactions precede binding of PRP31 to U4 snRNA. Knock-down of NUFIP leads to mislocalization of PRP31 and decreased association with U4. Moreover, NUFIP is associated with the SMN complex through direct interactions with Gemin3 and Gemin6. Altogether, our data suggest a model in which the NUFIP/R2TP system is connected with the SMN complex and facilitates assembly of U4 snRNP-specific proteins.     

A structural,phylogenetic, and functional study of 15.5-kD/Snu13 protein binding on U3 small nucleolar RNA

The 15.5-kD protein and its yeast homolog Snu13p bind U4 snRNA, U3 snoRNA, and the C/D box snoRNAs. In U4 snRNA, they associate with a helix-bulge-helix (K-turn) structure. U3 snoRNA contains two conserved pairs of boxes, C'/D and B/C, which were both expected to bind the 15.5-kD/Snu13 protein. Only binding to the B/C motif was experimentally demonstrated. Here, by chemical probing of in vitro reconstituted RNA/protein complexes, we demonstrate the independent binding of the 15.5-kD/Snu13 protein to each of the two motifs. Due to a highly reduced stem I (1 bp), the K-turn structure is not formed in the naked B/C motif. However, gel-shift experiments revealed a higher affinity of Snu13p for the B/C motif, compared to the C'/D motif. A phylogenetic analysis of U3 snoRNA, coupled with an analysis of Snu13p affinity for variant yeast C'/D and B/C motifs, and a study of the functionality of a truncated yeast U3 snoRNA carrying base substitutions in the C'/D and B/C motifs, revealed that conservation of the identities of residues 2 and 3 in the B/C K-turn is more important for Snu13p binding and U3 snoRNA function, than conservation of the identities of corresponding residues in the C'/D K-turn. This suggests that binding of Snu13p to K-turns with a very short helix I imposes sequence constraints in the bulge. Altogether, the data demonstrate the strong importance of the binding of the 15.5-kD/Snu13 protein to the C'/D and B/C motifs for both U3 snoRNP assembly and activity.     

Nutritional status modulates box C/D snoRNP biogenesis by regulated subcellular relocalization of the R2TP complex

Background

Box C/D snoRNPs, which are typically composed of box C/D snoRNA and the four core protein components Nop1, Nop56, Nop58, and Snu13, play an essential role in the modification and processing of pre-ribosomal RNA. The highly conserved R2TP complex, comprising the proteins Rvb1, Rvb2, Tah1, and Pih1, has been shown to be required for box C/D snoRNP biogenesis and assembly; however, the molecular basis of R2TP chaperone-like activity is not yet known.

Results

Here, we describe an unexpected finding in which the activity of the R2TP complex is required for Nop58 protein stability and is controlled by the dynamic subcellular redistribution of the complex in response to growth conditions and nutrient availability. In growing cells, the complex localizes to the nucleus and interacts with box C/D snoRNPs. This interaction is significantly reduced in poorly growing cells as R2TP predominantly relocalizes to the cytoplasm. The R2TP-snoRNP interaction is mainly mediated by Pih1.

Conclusions

The R2TP complex exerts a novel regulation on box C/D snoRNP biogenesis that affects their assembly and consequently pre-rRNA maturation in response to different growth conditions.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0404-4) contains supplementary material, which is available to authorized users.     

Analysis of Snu13p mutations reveals differential interactions with the U4 snRNA and U3 snoRNA

Pre-mRNA splicing is executed by the spliceosome, a complex of small nuclear RNAs (snRNAs) and numerous proteins. One such protein, 15.5K/Snu13p, is associated with the spliceosomal U4/U6.U5 tri-snRNP and box C/D small nucleolar ribonucleoprotein particles (snoRNPs), which act during preribosomal RNA (rRNA) processing. As such, it is the first splicing factor to be identified in two functionally distinct particles. 15.5K binds to an internal helix-bulge-helix (K-turn) structure in the U4 snRNA and two such structures in the U3 snoRNA. Previous work has concentrated on the structural basis of the interaction of 15.5K with the RNAs and has been carried out in vitro. Here we present a functional analysis of Snu13p in vivo, using a galactose inducible SNU13 strain to investigate the basis of three lethal mutations in Saccharomyces cerevisiae. Two are point mutations that map to the RNA-binding domain, and the third is a C-terminal deletion. These mutations result in accumulation of unspliced pre-mRNA, confirming a role for Snu13p in pre-mRNA splicing. In addition, these mutants also display rRNA processing defects that are variable in nature. Analysis of one mutant in the RNA-binding domain reveals a reduction in the levels of the U4 snRNA, U6 snRNA, and box C/D snoRNAs, but not H/ACA snoRNAs, supporting a role for Snu13p in accumulation and/or maintenance of specific RNAs. The mutations in the RNA-binding domain exhibit differential binding to the U4 snRNA and U3 snoRNA in vitro, suggesting that there are differences in the mode of interaction of Snu13p with these two RNAs.     

Accumulation of H/ACA snoRNPs depends on the integrity of the conserved central domain of the RNA-binding protein Nhp2p

Box H/ACA small nucleolar ribonucleoprotein particles (H/ACA snoRNPs) play key roles in the synthesis of eukaryotic ribosomes. How box H/ACA snoRNPs are assembled remains unknown. Here we show that yeast Nhp2p, a core component of these particles, directly binds RNA. In vitro, Nhp2p interacts with high affinity with RNAs containing irregular stem–loop structures but shows weak affinity for poly(A), poly(C) or for double-stranded RNAs. The central region of Nhp2p is believed to function as an RNA-binding domain, since it is related to motifs found in various RNA-binding proteins. Removal of two amino acids that shortens a putative β-strand element within Nhp2p central domain impairs the ability of the protein to interact with H/ACA snoRNAs in cell extracts. In vivo, this deletion prevents cell viability and leads to a strong defect in the accumulation of H/ACA snoRNAs and Gar1p. These data suggest that proper direct binding of Nhp2p to H/ACA snoRNAs is required for the assembly of H/ACA snoRNPs and hence for the stability of some of their components. In addition, we show that converting a highly conserved glycine residue (G₅₉) within Nhp2p central domain to glutamate significantly reduces cell growth at 30 and 37°C. Remarkably, this modification affects the steady-state levels of H/ACA snoRNAs and the strength of Nhp2p association with these RNAs to varying degrees, depending on the nature of the H/ACA snoRNA. Finally, we show that the modified Nhp2p protein whose interaction with H/ACA snoRNAs is impaired cannot accumulate in the nucleolus, suggesting that only the assembled H/ACA snoRNP particles can be efficiently retained in the nucleolus.     

Analysis of sequence and structural features that identify the B/C motif of U3 small nucleolar RNA as the recognition site for the Snu13p-Rrp9p protein pair

The eukaryal Snu13p/15.5K protein binds K-turn motifs in U4 snRNA and snoRNAs. Two Snu13p/15.5K molecules bind the nucleolar U3 snoRNA required for the early steps of preribosomal processing. Binding of one molecule on the C'/D motif allows association of proteins Nop1p, Nop56p, and Nop58p, whereas binding of the second molecule on the B/C motif allows Rrp9p recruitment. To understand how the Snu13p-Rrp9p pair recognizes the B/C motif, we first improved the identification of RNA determinants required for Snu13p binding by experiments using the systematic evolution of ligands by exponential enrichment. This demonstrated the importance of a U.U pair stacked on the sheared pairs and revealed a direct link between Snu13p affinity and the stability of helices I and II. Sequence and structure requirements for efficient association of Rrp9p on the B/C motif were studied in yeast cells by expression of variant U3 snoRNAs and immunoselection assays. A G-C pair in stem II, a G residue at position 1 in the bulge, and a short stem I were found to be required. The data identify the in vivo function of most of the conserved residues of the U3 snoRNA B/C motif. They bring important information to understand how different K-turn motifs can recruit different sets of proteins after Snu13p association.     

Box C/D small nucleolar RNA trafficking involves small nucleolar RNP proteins, nucleolar factors and a novel nuclear domain

Nucleolar localization of box C/D small nucleolar (sno) RNAs requires the box C/D motif and, in vertebrates, involves transit through Cajal bodies (CB). We report that in yeast, overexpression of a box C/D reporter leads to a block in the localization pathway with snoRNA accumulation in a specific sub-nucleolar structure, the nucleolar body (NB). The human survival of motor neuron protein (SMN), a marker of gems/CB, specifically localizes to the NB when expressed in yeast, supporting similarities between these structures. Box C/D snoRNA accumulation in the NB was decreased by mutation of Srp40 and increased by mutation of Nsr1p, two related nucleolar proteins that are homologous to human Nopp140 and nucleolin, respectively. Box C/D snoRNAs also failed to accumulate in the NB, and became delocalized to the nucleoplasm, upon depletion of any of the core snoRNP proteins, Nop1p/fibrillarin, Snu13p, Nop56p and Nop5p/Nop58p. We conclude that snoRNP assembly occurs either in the nucleoplasm, or during transit of snoRNAs through the NB, followed by routing of the complete snoRNP to functional sites of ribosome synthesis.     

Characterization of Saccharomyces cerevisiae Nop17p, a novel Nop58p-interacting protein that is involved in Pre-rRNA processing

In eukaryotes, pre-rRNA processing depends on cis-acting elements and on a large number of non-ribosomal trans-acting factors, including endonucleases and exonucleases, RNA helicases, rRNA modifying enzymes and components of snoRNPs. The exosome is a conserved eukaryotic protein complex containing multiple 3'-5' exonucleases, which has been implicated in pre-rRNA, snoRNA and snRNA processing, as well as in mRNA degradation. In order to identify new proteins involved in rRNA processing, we have screened a yeast two-hybrid cDNA library, to isolate proteins interacting with the exosome subunit Rrp43p. In this screen, a novel nucleolar protein, Nop17p, was identified which also interacts with the box C/D snoRNP protein Nop58p. The NOP17 gene is not essential for cell viability but its deletion causes a temperature-sensitive phenotype. Pre-rRNA processing analyses revealed that rRNA formation is affected in the Deltanop17 strain subjected to the non-permissive temperature, although it is not blocked completely. In addition, primer extension analyses of RNA isolated from Nop17p-depleted cells subjected to the non-permissive temperature indicates that the pre-rRNA is undergoing different modification or degradation processes in these cells as compared to the parental strain. Nop17p was recently described in the same complex as Nop58p and, interestingly, its depletion leads to mislocalization of Nop1p, Nop56p, Nop58p and Snu13p, which are the core proteins of the box C/D ribonucleoprotein (snoRNP), indicating that Nop17p function is required either for nucleolar retention or for the proper assembly of the box C/D snoRNP.     

AtNUFIP, an essential protein for plant development, reveals the impact of snoRNA gene organisation on the assembly of snoRNPs and rRNA methylation in Arabidopsis thaliana

In all eukaryotes, C/D small nucleolar ribonucleoproteins (C/D snoRNPs) are essential for methylation and processing of ribosomal RNAs. They consist of a box C/D small nucleolar RNA (C/D snoRNA) associated with four highly conserved nucleolar proteins. Recent data in HeLa cells and yeast have revealed that assembly of these snoRNPs is directed by NUFIP protein and other auxiliary factors. Nevertheless, the precise function and biological importance of NUFIP and the other assembly factors remains unknown. In plants, few studies have focused on RNA methylation and snoRNP biogenesis. Here, we identify and characterise the AtNUFIP gene that directs assembly of C/D snoRNP. To elucidate the function of AtNUFIP in planta, we characterized atnufip mutants. These mutants are viable but have severe developmental phenotypes. Northern blot analysis of snoRNA accumulation in atnufip mutants revealed a specific degradation of C/D snoRNAs and this situation is correlated with a reduction in rRNA methylation. Remarkably, the impact of AtNUFIP depends on the structure of snoRNA genes: it is essential for the accumulation of those C/D snoRNAs encoded by polycistronic genes, but not by monocistronic or tsnoRNA genes. We propose that AtNUFIP controls the kinetics of C/D snoRNP assembly on nascent precursors to overcome snoRNA degradation of aberrant RNPs. Finally, we show that AtNUFIP has broader RNP targets, controlling the accumulation of scaRNAs that direct methylation of spliceosomal snRNA in Cajal bodies.     

Archaeal ribosomal protein L7 is a functional homolog of the eukaryotic 15.5kD/Snu13p snoRNP core protein

Recent investigations have identified homologs of eukaryotic box C/D small nucleolar RNAs (snoRNAs) in Archaea termed sRNAs. Archaeal homologs of the box C/D snoRNP core proteins fibrillarin and Nop56/58 have also been identified but a homolog for the eukaryotic 15.5kD snoRNP protein has not been described. Our sequence analysis of archaeal genomes reveals that the highly conserved ribosomal protein L7 exhibits extensive homology with the eukaryotic 15.5kD protein. Protein binding studies demonstrate that recombinant Methanoccocus jannaschii L7 protein binds the box C/D snoRNA core motif with the same specificity and affinity as the eukaryotic 15.5kD protein. Identical to the eukaryotic 15.5kD core protein, archaeal L7 requires a correctly folded box C/D core motif and intact boxes C and D. Mutational analysis demonstrates that critical features of the box C/D core motif essential for 15.5kD binding are also required for L7 interaction. These include stem I which juxtaposes boxes C and D, as well as the sheared G:A pairs and protruded pyrimidine nucleotide of the asymmetric bulge region. The demonstrated presence of L7Ae in the Haloarcula marismortui 50S ribosomal subunit, taken with our demonstration of the ability of L7 to bind to the box C/D snoRNA core motif, indicates that this protein serves a dual role in Archaea. L7 functioning as both an sRNP core protein and a ribosomal protein could potentially regulate and coordinate sRNP assembly with ribosome biogenesis.     

Stable expression in yeast of the mature form of human telomerase RNA depends on its association with the box H/ACA small nucleolar RNP proteins Cbf5p, Nhp2p and Nop10p

Telomerase is a ribonucleoprotein (RNP) particle required for the replication of telomeres. The RNA component, termed hTR, of human telomerase contains a domain structurally and functionally related to box H/ACA small nucleolar RNAs (snoRNAs). Furthermore, hTR is known to be associated with two core components of H/ACA snoRNPs, hGar1p and Dyskerin (the human counterpart of yeast Cbf5p). To assess the functional importance of the association of hTR with H/ACA snoRNP core proteins, we have attempted to express hTR in a genetically tractable system, Saccharomyces cerevisiae. Both mature non-polyadenylated and polyadenylated forms of hTR accumulate in yeast. The former is associated with all yeast H/ACA snoRNP core proteins, unlike TLC1 RNA, the endogenous RNA component of yeast telomerase. We show that the presence of the H/ACA snoRNP proteins Cbf5p, Nhp2p and Nop10p, but not Gar1p, is required for the accumulation of mature non-polyadenylated hTR in yeast, while accumulation of TLC1 RNA is not affected by the absence of any of these proteins. Our results demonstrate that yeast telomerase is unrelated to H/ACA snoRNPs. In addition, they show that the accumulation in yeast of the mature RNA component of human telomerase depends on its association with three of the four core H/ACA snoRNP proteins. It is likely that this is the case in human cells as well.     

The Shq1p.Naf1p complex is required for box H/ACA small nucleolar ribonucleoprotein particle biogenesis

Small nucleolar ribonucleoprotein particles (snoRNPs) are essential cofactors in ribosomal RNA metabolism. Although snoRNP composition has been thoroughly characterized, the biogenesis process of these particles is poorly understood. We have identified two proteins from the yeast Saccharomyces cerevisiae, Yil104c/Shq1p and Ynl124w/Naf1p, which are essential and required for the stability of box H/ACA snoRNPs. Depletion of either Shq1p or Naf1p leads to a dramatic and specific decrease in box H/ACA snoRNA levels in vivo. A severe concomitant defect in ribosomal RNA processing is observed, consistent with the depletion of this family of snoRNAs. Shq1p and Naf1p show nuclear localization and interact with Nhp2p and Cbf5p, two core proteins of mature box H/ACA snoRNPs. Shq1p and Naf1p form a complex, but they are not strongly associated with box H/ACA snoRNPs. We propose that Shq1p and Naf1p are involved in the early biogenesis steps of box H/ACA snoRNP assembly.     

Cbf5p,a potential pseudouridine synthase,and Nhp2p,a putative RNA-binding protein,are present together with Gar1p in all H BOX/ACA-motif snoRNPs and constitute a common bipartite structure.

The eukaryotic nucleolus contains a large number of small nucleolar RNAs (snoRNAs) that are involved in preribosomal RNA (pre-rRNA) processing. The H box/ACA-motif (H/ACA) class of snoRNAs has recently been demonstrated to function as guide RNAs targeting specific uridines in the pre-rRNA for pseudouridine (psi) synthesis. To characterize the protein components of this class of snoRNPs, we have purified the snR42 and snR30 snoRNP complexes by anti-m3G-immunoaffinity and Mono-Q chromatography of Saccharomyces cerevisiae extracts. Sequence analysis of the individual polypeptides demonstrated that the three proteins Gar1p, Nhp2p, and Cbf5p are common to both the snR30 and snR42 complexes. Nhp2p is a highly basic protein that belongs to a family of putative RNA-binding proteins. Cbf5p has recently been demonstrated to be involved in ribosome biogenesis and also shows striking homology with known prokaryotic psi synthases. The presence of Cbf5p, a putative psi synthase in each H/ACA snoRNP suggests that this class of RNPs functions as individual modification enzymes. Immunoprecipitation studies using either anti-Cbf5p antibodies or a hemagglutinin-tagged Nhp2p demonstrated that both proteins are associated with all H/ACA-motif snoRNPs. In vivo depletion of Nhp2p results in a reduction in the steady-state levels of all H/ACA snoRNAs. Electron microscopy of purified snR42 and snR30 particles revealed that these two snoRNPs possess a similar bipartite structure that we propose to be a major structural determining principle for all H/ACA snoRNPs.     

Interaction of Saccharomyces Cdc13p with Pol1p, Imp4p, Sir4p and Zds2p is involved in telomere replication, telomere maintenance and cell growth control

Telomeres are the physical ends of eukaryotic chromosomes. They are important for maintaining the integrity of chromosomes and this function is mediated through a number of protein factors. In Saccharomyces cerevisiae, Cdc13p binds to telomeres and affects telomere maintenance, telomere position effects and cell cycle progression through G₂/M phase. We identified four genes encoding Pol1p, Sir4p, Zds2p and Imp4p that interact with amino acids 1–252 of Cdc13p using a yeast two-hybrid screening system. Interactions of these four proteins with Cdc13p were through direct protein–protein interactions as judged by in vitro pull-down assays. Direct protein–protein interactions were also observed between Pol1p–Imp4p, Pol1p–Sir4p and Sir4p–Zds2p, whereas no interaction was detected between Imp4p–Sir4p and Zds2p–Imp4p, suggesting that protein interactions were specific in the complex. Pol1p was shown to interact with Cdc13p. Here we show that Zds2p and Imp4p also form a stable complex with Cdc13p in yeast cells, because Zds2p and Imp4p co-immunoprecipitate with Cdc13p, whereas Sir4p does not. The function of the N-terminal 1–252 region of Cdc13p was also analyzed. Expressing Cdc13(252–924)p, which lacks amino acids 1–252 of Cdc13p, causes defects in progressive cell growth and eventually arrested in the G₂/M phase of the cell cycle. These growth defects were not caused by progressive shortening of telomeres because telomeres in these cells were long. Point mutants in the amino acids 1–252 region of Cdc13p that reduced the interaction between Cdc13p and its binding proteins resulted in varying level of defects in cell growth and telomeres. These results indicate that the interactions between Cdc13(1–252)p and its binding proteins are important for the function of Cdc13p in telomere regulation and cell growth. Together, our results provide evidence for the formation of a Cdc13p-mediated telosome complex through its N-terminal region that is involved in telomere maintenance, telomere length regulation and cell growth control.