Insights into mRNP biogenesis provided by new genetic interactions among export and transcription factors.

BACKGROUND: The various steps of mRNP biogenesis (transcription, processing and export) are interconnected. It has been shown that the transcription machinery plays a pivotal role in mRNP assembly, since several mRNA export factors are recruited during transcription and physically interact with components of the transcription machinery. Although the shuttling DEAD-box protein Dbp5p is concentrated on the cytoplasmic fibrils of the NPC, previous studies demonstrated that it interacts physically and genetically with factors involved in transcription initiation. RESULTS: We investigated the effect of mutations affecting various components of the transcription initiation apparatus on the phenotypes of mRNA export mutant strains. Our results show that growth and mRNA export defects of dbp5 and mex67 mutant strains can be suppressed by mutation of specific transcription initiation components, but suppression was not observed for mutants acting in the very first steps of the pre-initiation complex (PIC) formation. CONCLUSIONS: Our results indicate that mere reduction in the amount of mRNP produced is not sufficient to suppress the defects caused by a defective mRNA export factor. Suppression occurs only with mutants affecting events within a narrow window of the mRNP biogenesis process. We propose that reducing the speed with which transcription converts from initiation and promoter clearance to elongation may have a positive effect on mRNP formation by permitting more effective recruitment of partially-functional mRNP proteins to the nascent mRNP.

Microarray analysis of cytoplasmic versus whole cell RNA reveals a considerable number of missed and false positive mRNAs.

With no known exceptions, every published microarray study to determine differential mRNA levels in eukaryotes used RNA extracted from whole cells. It is assumed that the use of whole cell RNA in microarray gene expression analysis provides a legitimate profile of steady-state mRNA. Standard labeling methods and the prevailing dogma that mRNA resides almost exclusively in the cytoplasm has led to the long-standing belief that the nuclear RNA contribution is negligible. We report that unadulterated cytoplasmic RNA uncovers differentially expressed mRNAs that otherwise would not have been detected when using whole cell RNA and that the inclusion of nuclear RNA has a large impact on whole cell gene expression microarray results by distorting the mRNA profile to the extent that a substantial number of false positives are generated. We conclude that to produce a valid profile of the steady-state mRNA population, the nuclear component must be excluded, and to arrive at a more realistic view of a cell's gene expression profile, the nuclear and cytoplasmic RNA fractions should be analyzed separately.

Synthetic genetic array analysis in Saccharomyces cerevisiae provides evidence for an interaction between RAT8/DBP5 and genes encoding P-body components.

Coordination of the multiple steps of mRNA biogenesis helps to ensure proper regulation of gene expression. The Saccharomyces cerevisiae DEAD-box protein Rat8p/Dbp5p is an essential mRNA export factor that functions at the nuclear pore complex (NPC) where it is thought to remodel mRNA/protein complexes during mRNA export. Rat8p also functions in translation termination and has been implicated in functioning during early transcription. We conducted a synthetic genetic array analysis (SGA) using a strain harboring the temperature-sensitive rat8-2 allele. Although RAT8 had been shown to interact genetically with >15 other genes, we identified >40 additional genes whose disruption in a rat8-2 background causes synthetic lethality or dramatically reduced growth. Included were five that encode components of P-bodies, sites of cytoplasmic mRNA turnover and storage. Wild-type Rat8p localizes to NPCs and diffusely throughout the cell but rat8-2p localized to cytoplasmic granules at nonpermissive temperature that are distinct from P-bodies. In some genetic backgrounds, these granules also contain poly(A)-binding protein, Pab1p, and additional mRNA export factors. Although these foci are distinct from P-bodies, the two merge under heat-stress conditions. We suggest that these granules reflect defective mRNP remodeling during mRNA export and during cytoplasmic mRNA metabolism.

Coupling of termination, 3' processing, and mRNA export.

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.

Altered MicroRNA expression confined to specific epithelial cell subpopulations in breast cancer.

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.