Dissociation of Rpb4 from RNA polymerase II is important for yeast functionality.

Rpb4 is an RNA polymerase II (Pol II) subunit that binds Pol II transcripts co-transcriptionally, accompanies them to the cytoplasm and modulates mRNA export, translation and decay by interacting with cytoplasmic RNA modulators. The importance of the cytoplasmic roles of Rpb4 was challenged by a study reporting that the phenotype of rpb2Δ rpb4Δ cells can be rescued by an Rpb2-Rpb4 fusion protein, assuming that its Rpb4 moiety cannot dissociate from Pol II and functions in the cytoplasm. Here we demonstrate that although the fusion protein supports normal transcription, it adversely affects mRNA decay, cell proliferation and adaptability-e.g., response to stress. These defects are similar, albeit milder, than the defects that characterize rpb4Δ cells. At least two mechanisms alleviate the deleterious effect of the fusion protein. First, a portion of this fusion protein is cleaved into free Rpb2 and Rpb4. The free Rpb4 is functional, as it binds mRNAs and polysomes, like WT Rpb4. Second, the fusion protein is also capable of binding poly(A)+ mRNAs in the cytoplasm, in an Rpb7-mediated manner, probably complementing the functions of the diminished Rpb4. Collectively, normal coupling between mRNA synthesis and decay requires wild-type configuration of Rpb4, and fusing Rpb4 to Rpb2 compromises this coupling.

Pheromone-encoding mRNA is transported to the yeast mating projection by specific RNP granules.

Association of messenger RNAs with large complexes such as processing bodies (PBs) plays a pivotal role in regulating their translation and decay. Little is known about other possible functions of these assemblies. Exposure of haploid yeast cells, carrying mating type "a," to "α pheromone" stimulates polarized growth resulting in a "shmoo" projection; it also induces synthesis of "a pheromone," encoded by MFA2. In this paper, we show that, in response to α pheromone, MFA2 mRNA is assembled with two types of granules; both contain some canonical PB proteins, yet they differ in size, localization, motility, and sensitivity to cycloheximide. Remarkably, one type is involved in mRNA transport to the tip of the shmoo, whereas the other-in local translation in the shmoo. Normal assembly of these granules is critical for their movement, localization, and for mating. Thus, MFA2 mRNAs are transported to the shmoo tip, in complex with PB-like particles, where they are locally translated.

Promoter elements regulate cytoplasmic mRNA decay.

Promoters are DNA elements that enable transcription and its regulation by trans-acting factors. Here, we demonstrate that yeast promoters can also regulate mRNA decay after the mRNA leaves the nucleus. A conventional yeast promoter consists of a core element and an upstream activating sequence (UAS). We find that changing UASs of a reporter gene without altering the transcript sequence affects the transcript's decay kinetics. A short cis element, comprising two Rap1p-binding sites, and Rap1p itself, are necessary and sufficient to induce enhanced decay of the reporter mRNA. Furthermore, Rap1p stimulates both the synthesis and the decay of a specific population of endogenous mRNAs. We propose that Rap1p association with target promoter in the nucleus affects the composition of the exported mRNP, which in turn regulates mRNA decay in the cytoplasm. Thus, promoters can play key roles in determining mRNA levels and have the capacity to coordinate rates of mRNA synthesis and decay.

RNA polymerase II subunits link transcription and mRNA decay to translation.

Little is known about crosstalk between the eukaryotic transcription and translation machineries that operate in different cell compartments. The yeast proteins Rpb4p and Rpb7p represent one such link as they form a heterodimer that shuttles between the nucleus, where it functions in transcription, and the cytoplasm, where it functions in the major mRNA decay pathways. Here we show that the Rpb4/7 heterodimer interacts physically and functionally with components of the translation initiation factor 3 (eIF3), and is required for efficient translation initiation. Efficient translation in the cytoplasm depends on association of Rpb4/7 with RNA polymerase II (Pol II) in the nucleus, leading to a model in which Pol II remotely controls translation. Hence, like in prokaryotes, the eukaryotic translation is coupled to transcription. We propose that Rpb4/7, through its interactions at each step in the mRNA lifecycle, represents a class of factors, "mRNA coordinators," which integrate the various stages of gene expression into a system.

The Rpb7p subunit of yeast RNA polymerase II plays roles in the two major cytoplasmic mRNA decay mechanisms.

The steady-state level of mRNAs is determined by the balance between their synthesis by RNA polymerase II (Pol II) and their decay. In the cytoplasm, mRNAs are degraded by two major pathways; one requires decapping and 5' to 3' exonuclease activity and the other involves 3' to 5' degradation. Rpb7p is a Pol II subunit that shuttles between the nucleus and the cytoplasm. Here, we show that Rpb7p is involved in the two mRNA decay pathways and possibly couples them. Rpb7p stimulates the deadenylation stage required for execution of both pathways. Additionally, Rpb7p is both an active component of the P bodies, where decapping and 5' to 3' degradation occur, and is capable of affecting the P bodies function. Moreover, Rpb7p interacts with the decapping regulator Pat1p in a manner important for the mRNA decay machinery. Rpb7p is also involved in the second pathway, as it stimulates 3' to 5' degradation. Our genetic analyses suggest that Rpb7p plays two distinct roles in mRNA decay, which can both be uncoupled from Rpb7p's role in transcription. Thus, Rpb7p plays pivotal roles in determining mRNA levels.

Infection stages of the dermatophyte pathogen Trichophyton: microscopic characterization and proteolytic enzymes.

Dermatophytes are pathogenic fungi that infect human skin, nails and hair and cause dermatophytosis. Trichophyton mentagrophytes is one of the most widespread species that belong to this group. Infection of the skin tissues include several stages, i.e., adhesion to the surface of the skin, invasion into the sublayers by the penetration of fungal elements and secretion of enzymes that degrade the skin components. In this study we have followed the morphology of the fungal elements, such as arthroconidia and hyphae, during the adhesion and invasion stages. Skin explants were inoculated with the dermatophyte and observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Skin explants were also inoculated with a transgenic isolate of T. mentagrophytes expressing the green fluorescent protein (GFP). The infected sublayers were investigated by confocal scanning laser microscopy (CSLM). As an adaptation to the tissue environment, the dermatophyte produced long fibrils when it is on the open surface of the stratum corneum, while short and thin fibrils are produced inside the dense sublayers. The short and long projections might have a role in adhesion. Invasion may be produced by mechanical and biochemical means. Invasion of the tissue showed hyphal branching and growth in multiple directions. The proteolytic profile was assayed by substrate gel and proteolytic activity. Two serine proteases of similar molecular weight were secreted during growth on the epidermal matrix components keratin and elastin. The dermatophyte may use the proteolytic enzymes to invade the surface and also the deep layer of the skin in immunocompromised patients. Dermatophytes, which are well adapted infectious agents, seem to use their mechanical and biochemical capabilities to invade the skin tissue effectively.

Nucleocytoplasmic shuttling of the Rpb4p and Rpb7p subunits of Saccharomyces cerevisiae RNA polymerase II by two pathways.

Rpb4p and Rpb7p are subunits of the RNA polymerase II of Saccharomyces cerevisiae that form a dissociable heterodimeric complex. Whereas the only reported function of Rpb7p is related to transcription, Rpb4p has been found to also act in mRNA export and in the major mRNA decay pathway that operates in the cytoplasm, thus raising the possibility that Rpb4p links between the nuclear and cytoplasmic processes. Here we show that both Rpb4p and Rpb7p shuttle between the nucleus and the cytoplasm. Shuttling kinetics of the two proteins are similar as long as their interaction is possible, suggesting that they shuttle as a heterodimer. Under normal conditions, shuttling of Rpb4p and Rpb7p depends on ongoing transcription. However, during severe stresses of heat shock, ethanol, and starvation, the two proteins shuttle via a transcription-independent pathway. Thus, Rpb4p and Rpb7p shuttle via two pathways, depending on environmental conditions.

The RNA polymerase II subunit Rpb4p mediates decay of a specific class of mRNAs.

It is commonly appreciated that the mRNA level is determined by the balance between its synthetic and decay kinetics. Yet, little is known about coordination between these distinct processes. A major pathway of the eukaryotic mRNA decay initiates with shortening of the mRNA poly(A) tail (deadenylation), followed by removal of the mRNA 5' cap structure and its subsequent exonucleolytic degradation. Here we report that a subunit of RNA polymerase II, Rpb4p, is required for the decay of a class of mRNAs whose products are involved in protein synthesis. Cells lacking RPB4 are defective in the deadenylation and post-deadenylation steps of representatives of this class of mRNAs. Moreover, Rpb4p interacts with both the mRNP and with subunits of the mRNA decay complex Pat1/Lsm1-7 that enhances decapping. Consistently, a portion of Rpb4p is localized in P bodies, where mRNA decapping and degradation is executed, and mutations in RPB4 increase the number of P bodies per cell. We propose that Rpb4p has a dual function in mRNA decay. It promotes or enhances the deadenylation process of specific mRNAs and recruits Pat1/Lsm1-7 to these mRNAs, thus stimulating their decapping and further decay. In this way, Rpb4p might link the activity of the basal transcription apparatus with that of the mRNA decay machinery.