MicroRNAs (miRNAs) are a new class of short noncoding regulatory RNAs (18-25 nucleotides) that are involved in diverse developmental and pathologic processes. Altered miRNA expression has been associated with several types of human cancer. However, most studies did not establish whether miRNA expression changes occurred within cells undergoing malignant transformation. To obtain insight into miRNA deregulation in breast cancer, we implemented an in situ hybridization (ISH) method to reveal the spatial distribution of miRNA expression in archived formalin-fixed, paraffin-embedded specimens representing normal and tumor tissue from >100 patient cases. Here, we report that expression of miR-145 and miR-205 was restricted to the myoepithelial/basal cell compartment of normal mammary ducts and lobules, whereas their accumulation was reduced or completely eliminated in matching tumor specimens. Conversely, expression of other miRNAs was detected at varying levels predominantly within luminal epithelial cells in normal tissue; expression of miR-21 was frequently increased, whereas that of let-7a was decreased in malignant cells. We also analyzed the association of miRNA expression with that of epithelial markers; prognostic indicators such as estrogen receptor, progesterone receptor, and HER2; as well as clinical outcome data. This ISH approach provides a more direct and informative assessment of how altered miRNA expression contributes to breast carcinogenesis compared with miRNA expression profiling in gross tissue biopsies. Most significantly, early manifestation of altered miR-145 expression in atypical hyperplasia and carcinoma in situ lesions suggests that this miRNA may have a potential clinical application as a novel biomarker for early detection.

• Abstract. To identify genes involved in the export of messenger RNA from the nucleus to the cytoplasm, we used an in situ hybridization assay to screen temperature-sensitive strains of Saccharomyces cerevisiae. This identified those which accumulated poly(A) ÷ RNA in their nuclei when shifted to the non-permissive temperature of 37°C. We describe here the properties of yeast strains carrying mutations in the RA T2 gene (RA Tribonucleic acid trafficking) and the cloning of the RA T2 gene. Only a low percentage of cells carrying the rat2-1 allele showed nuclear accumulation of poly(A) + RNA when cultured at 15 ° or 23°C, but within 4 h of a shift to the nonpermissive temperature of 37°C, poly(A) ÷ RNA accumulated within the nuclei of approximately 80% of cells. No defect was seen in the nuclear import of a reporter protein bearing a nuclear localization signal. Nuclear pore complexes (NPCs) are distributed relatively evenly around the nuclear envelope in wild-type cells. In cells carrying either the rat2-1 or rat2-2 allele, NPCs were clustered together into one or a few regions of the nuclear envelope. This clustering was a constitutive property of mutant cells. NPCs remained clustered in crude nuclei isolated from mutant cells, indicating that these clusters are not able to redistribute around the nuclear envelope when nuclei are separated from cytoplasmic components. Electron microscopy revealed that these clusters were frequently found in a protuberance of the nuclear envelope and were often located close to the spindle pole body. The RA T2 gene encodes a 120-kD protein without similarity to other known proteins. It was essential for growth only at 37°C, but the growth defect at high temperature could be suppressed by growth of mutant ceils in the presence of high osmolarity media containing 1.0 M sorbitol or 0.9 M NaC1. The phenotypes seen in cells carrying a disruption of the RA T2 gene were very similar to those seen with the rat2-1 and rat2-2 alleles. Epitope tagging was used to show that Rat2p is located at the nuclear periphery and co-localizes with yeast NPC proteins recognized by the RL1 monoclonal antibody. The rat2-1 allele was synthetically lethal with both the rat3-1/nup133-1 and rat7-1/nup159-1 alleles. These results indicate that the product of this gene is a nucleoporin which we refer to as Rat2pfNupl20p.

We reported previously that heat or ethanol shock in Saccharomyces cerevisiae leads to nuclear retention of most poly(A) + RNA but heat shock mRNAs (encoding Hsp70 proteins Ssa1p and Ssa4p) are efficiently exported in a process that is independent of the small GTPase Ran/Gsp1p, which is essential for most nucleocytoplasmic transport. To gain further insights into proteins essential or nonessential for export of heat shock mRNAs, in situ hybridization analyses to detect mRNA and pulse-labeling of proteins were used to examine several yeast mutant strains for their ability to export heat shock mRNAs following stress. Rip1p is a 42-kD protein associated with nuclear pore complexes and contains nucleoporin-like repeat sequences. It is dispensable for growth of yeast cells under normal conditions, but we report that it is essential for the export of heat shock mRNAs following stress. When SSA4 mRNA was induced from a GAL promoter in the absence of stress, it was efficiently exported in a strain lacking RIP1, indicating that Rip1p is required for export of heat shock mRNAs only following stress. Npl3p, a key mediator of export of poly(A) + RNA, was not required for heat shock mRNA export, whereas Rss1p/Gle1p, a NES-containing factor essential for poly(A) + RNA export, was also required for export of heat shock mRNAs after stress. High-level expression of the HIV-1 Rev protein, but not of Rev mutants, led to a partial block in export of heat shock mRNAs following stress. The data suggest a model wherein the requirement for Npl3p defines the mRNA export pathway, the requirement for Rip1p defines a pathway used for export of heat shock mRNAs after stress, and additional factors, including Rss1p/Gle1p and several nucleoporins (Rat7p/Nup159p, Rat2p/Nup120p, and Nup145p/Rat10p), are required in both pathways.[Key Words: RNA export; heat shock; RIP1; hnRNP; RSS1/GLE1; Rev]Received July 1, 1997; revised version accepted August 28, 1997.A distinguishing feature of eukaryotic cells is the nucleus, a distinct subcellular compartment separated from the cytoplasm by the double-membraned nuclear envelope. Embedded within the nuclear envelope are nuclear pore complexes (NPCs) that serve as the only known channels for transport between the nucleus and the cytoplasm (for review, see Davis 1995;Panté and Aebi 1996). Transport of macromolecules through NPCs is signal-mediated, saturable, and energy dependent. Considerable progress has been made in recent years in identifying (1) receptor molecules that recognize nuclear localization signals (NLSs) within karyophilic proteins and mediate interactions between these proteins and NPCs, (2) a small Ras-like GTPase (Ran in metazoan cells and Gsp1p/Gsp2p in Saccharomyces cerevisiae) and its accessory proteins, which play a central role in nuclear protein import, and (3) distinct components of NPCs required for nuclear protein import (for review, see Gö rlich and

In a screen to identify genes required for mRNA export in Saccharomyces cerevisiae, we isolated an allele of poly(A) polymerase (PAP1) and novel alleles encoding several other 3 processing factors. Many newly isolated and some previously described mutants (rna14-48, rna14-49, rna14-64, rna15-58, and pcf11-1 strains) are defective in polymerase II (Pol II) termination but, interestingly, retain the ability to polyadenylate these improperly processed transcripts at the nonpermissive temperature. Deletion of the cis-acting sequences required to couple 3 processing and termination also produces transcripts that fail to exit the nucleus, suggesting that all of these processes (cleavage, termination, and export) are coupled. We also find that several but not all mRNA export mutants produce improperly 3 processed transcripts at the nonpermissive temperature. 3 maturation defects in mRNA export mutants include improper Pol II termination and/or the previously characterized hyperpolyadenylation of transcripts. Importantly, not all mRNA export mutants have defects in 3 processing. The similarity of the phenotypes of some mRNA export mutants and 3 processing mutants indicates that some factors from each process may mechanistically interact to couple mRNA processing and export. Consistent with this assumption, we present evidence that Xpo1p interacts in vivo with several 3 processing factors and that the addition of recombinant Xpo1p to in vitro processing reaction mixtures stimulates 3 maturation. Of the core 3 processing factors tested (Rna14p, Rna15p, Pcf11p, Hrp1p, Fip1p, and Cft1p), only Hrp1p shuttles. Overexpression of Rat8p/Dbp5p suppresses both 3 processing and mRNA export defects found in xpo1-1 cells.One of the defining features of eukaryotic cells is the physical separation of the nucleus, where mRNAs are synthesized, from the cytoplasm, where protein synthesis occurs. Gene expression and cell function require the efficient transport of macromolecules between these two compartments. All exchange between the nucleus and cytoplasm takes place through nuclear pore complexes (NPCs) that perforate the nuclear envelope and permit selective passage in both directions of molecules and complexes containing transport signals. Interactions between the import substrate or complex and NPC are mediated by import or export receptors that bind directly to specific transport signals on the substrate and NPCs. Directionality of this process is imparted through the activity of a small GTPase (Gsp1p in Saccharomyces cerevisiae and Ran in metazoans) that modulates the receptor's affinity for the substrate on opposite sides of the nuclear envelope.Export of mRNA to the cytoplasm appears to be more complex than transport of proteins. mRNAs are exported as messenger ribonucleoprotein complexes (mRNPs) whose assembly begins during transcription (27,55). mRNA biogenesis requires multiple processing steps including 5Ј capping, splicing, and 3Ј cleavage or polyadenylation. Most of these reactions are accomplished during transcription by se...

To identify genes whose products play potential roles in the nucleocytoplasmic export of messenger RNA, we isolated temperature-sensitive strains of Saccharomyces cerevisiae and examined them by fluorescent in situ hybridization. With the use of a digoxigen-tagged oligo-(dT)50 probe, we identified those that showed nuclear accumulation of poly(A)+ RNA when cells were shifted to the nonpermissive temperature. We describe here the properties of yeast strains bearing the rat3-1 mutation (RAT -ribonucleic acid trafficking) and the cloning of the RAT3 gene. When cultured at the permissive temperature of 23°C, fewer than 10% of cells carrying the rat3-1 allele showed nuclear accumulation of poly(A)+ RNA, whereas approximately 70% showed nuclear accumulation of poly(A)+ RNA after a shift to 37°C for 4 h. In wild-type cells, nuclear pore complexes (NPCs) are distributed relatively evenly around the nuclear envelope. Both indirect immunofluorescence analysis and electron microscopy of rat3-1 cells indicated that NPCs were clustered into one or a few regions of the NE in mutant cells. Similar NPC clustering was seen in mutant cells cultured at temperatures between 15°C and 37°C. The RAT3 gene encodes an 1157-amino acid protein without similarity to other known proteins. It is essential for growth only at 37°C. Cells carrying a disruption of the RAT3 gene were very similar to cells carrying the original rat3-1 mutation; they showed temperature-dependent nuclear accumulation of poly(A)+ RNA and exhibited constitutive clustering of NPCs. Epitope tagging of Rat3p demonstrated that it is located at the nuclear periphery and co-localizes with nuclear pore proteins recognized by the RL1 monoclonal antibody. We refer to this nucleoporin as Rat3p/Nupl33p.