Genes with small open reading frames (sORFs; <100 amino acids) represent an untapped source of important biology. sORFs largely escaped analysis because they were difficult to predict computationally and less likely to be targeted by genetic screens. Thus, the substantial number of sORFs and their potential importance have only recently become clear. To investigate sORF function, we undertook the first functional studies of sORFs in any system, using the model eukaryote Saccharomyces cerevisiae. Based on independent experimental approaches and computational analyses, evidence exists for 299 sORFs in the S. cerevisiae genome, representing ∼5% of the annotated ORFs. We determined that a similar percentage of sORFs are annotated in other eukaryotes, including humans, and 184 of the S. cerevisiae sORFs exhibit similarity with ORFs in other organisms. To investigate sORF function, we constructed a collection of gene-deletion mutants of 140 newly identified sORFs, each of which contains a strain-specific “molecular barcode,” bringing the total number of sORF deletion strains to 247. Phenotypic analyses of the new gene-deletion strains identified 22 sORFs required for haploid growth, growth at high temperature, growth in the presence of a nonfermentable carbon source, or growth in the presence of DNA damage and replication-arrest agents. We provide a collection of sORF deletion strains that can be integrated into the existing deletion collection as a resource for the yeast community for elucidating gene function. Moreover, our analyses of the S. cerevisiae sORFs establish that sORFs are conserved across eukaryotes and have important biological functions.

Aphysical and functional link between the nuclear pore complex (NPC) and the spindle checkpoint machinery has been established in the yeast Saccharomyces cerevisiae. We show that two proteins required for the execution of the spindle checkpoint, Mad1p and Mad2p, reside predominantly at the NPC throughout the cell cycle. There they are associated with a subcomplex of nucleoporins containing Nup53p, Nup170p, and Nup157p. The association of the Mad1p–Mad2p complex with the NPC requires Mad1p and is mediated in part by Nup53p. On activation of the spindle checkpoint, we detect changes in the interactions between these proteins, including the release of Mad2p (but not Mad1p) from the NPC and the accumulation of Mad2p at kinetochores. Accompanying these events is the Nup53p-dependent hyperphosphorylation of Mad1p. On the basis of these results and genetic analysis of double mutants, we propose a model in which Mad1p bound to a Nup53p-containing complex sequesters Mad2p at the NPC until its release by activation of the spindle checkpoint. Furthermore, we show that the association of Mad1p with the NPC is not passive and that it plays a role in nuclear transport.

Chromosomal instability (CIN) is a hallmark of many cancers and a major contributor to tumorigenesis. Centromere and kinetochore associated proteins such as the evolutionarily conserved centromeric histone H3 variant CENP-A, associate with centromeric DNA for centromere function and chromosomal stability. Stringent regulation of cellular CENP-A levels prevents its mislocalization in yeast and flies to maintain genome stability. CENP-A overexpression and mislocalization are observed in several cancers and reported to be associated with increased invasiveness and poor prognosis. We examined whether there is a direct relationship between mislocalization of overexpressed CENP-A and CIN using HeLa and chromosomally stable diploid RPE1 cell lines as model systems. Our results show that mislocalization of overexpressed CENP-A to chromosome arms leads to chromosome congression defects, lagging chromosomes, micronuclei formation and a delay in mitotic exit. CENP-A overexpressing cells showed altered localization of centromere and kinetochore associated proteins such as CENP-C, CENP-T and Nuf2 leading to weakened native kinetochores as shown by reduced interkinetochore distance and CIN. Importantly, our results show that mislocalization of CENP-A to chromosome arms is one of the major contributors for CIN as depletion of histone chaperone DAXX prevents CENP-A mislocalization and rescues the reduced interkinetochore distance and CIN phenotype in CENP-A overexpressing cells. In summary, our results establish that CENP-A overexpression and mislocalization result in a CIN phenotype in human cells. This study provides insights into how overexpression of CENP-A may contribute to CIN in cancers and underscore the importance of understanding the pathways that prevent CENP-A mislocalization for genome stability.

Analysis of global gene expression in Saccharomyces cerevisiae by the serial analysis of gene expression technique has permitted the identification of at least 302 previously unidentified transcripts from nonannotated open reading frames (NORFs). Transcription of one of these, NORF5/HUG1 (hydroxyurea and UV and gamma radiation induced), is induced by DNA damage, and this induction requires MEC1, a homolog of the ataxia telangiectasia mutated (ATM) gene. DNA damage-specific induction of HUG1, which is independent of the cell cycle stage, is due to the alleviation of repression by the Crt1p-Ssn6p-Tup1p complex. Overexpression of HUG1 is lethal in combination with a mec1 mutation in the presence of DNA damage or replication arrest, whereas a deletion of HUG1 rescues the lethality due to a mec1 null allele. HUG1 is the first example of a NORF with important biological functional properties and defines a novel component of the MEC1checkpoint pathway.

The kinetochore (centromeric DNA and associated proteins) is a key determinant for high fidelity chromosome transmission. Evolutionarily conserved Scm3p is an essential component of centromeric chromatin and is required for assembly and function of kinetochores in humans, fission yeast, and budding yeast. Overexpression of HJURP, the mammalian homolog of budding yeast Scm3p, has been observed in lung and breast cancers and is associated with poor prognosis; however, the physiological relevance of these observations is not well understood. We overexpressed SCM3 and HJURP in Saccharomyces cerevisiae and HJURP in human cells and defined domains within Scm3p that mediate its chromosome loss phenotype. Our results showed that the overexpression of SCM3 (GALSCM3) or HJURP (GALHJURP) caused chromosome loss in a wild-type yeast strain, and overexpression of HJURP led to mitotic defects in human cells. GALSCM3 resulted in reduced viability in kinetochore mutants, premature separation of sister chromatids, and reduction in Cse4p and histone H4 at centromeres. Overexpression of CSE4 or histone H4 suppressed chromosome loss and restored levels of Cse4p at centromeres in GALSCM3 strains. Using mutant alleles of scm3, we identified a domain in the N-terminus of Scm3p that mediates its interaction with CEN DNA and determined that the chromosome loss phenotype of GALSCM3 is due to centromeric association of Scm3p devoid of Cse4p/H4. Furthermore, we determined that similar to other systems the centromeric association of Scm3p is cell cycle regulated. Our results show that altered stoichiometry of Scm3p/HJURP, Cse4p, and histone H4 lead to defects in chromosome segregation. We conclude that stringent regulation of HJURP and SCM3 expression are critical for genome stability. Proper chromosome segregation is essential for normal cell proliferation. Segregation errors lead to aneuploidy, a direct cause of birth defects and a hallmark of cancer. The kinetochore (centromeric DNA and associated proteins) is one of the key determinants for faithful chromosome transmission. Misregulation of kinetochore proteins such as HJURP has been observed in various cancers, however the biological relevance of this observation is not well understood. We determined that altered dosage of HJURP and its budding yeast homolog SCM3 leads to defects in chromosome segregation in yeast and human cells. We identified the centromeric DNA–interacting domain of Scm3p and determined that association of Scm3p devoid of Cse4p leads to chromosome segregation defects. Our findings suggest that stringent regulation of Scm3p/HJURP, Cse4p, and histone H4 is critical for maintenance of genome stability.