Efficient PCR-based gene targeting in isolates of the nonconventional yeast Debaryomyces hansenii.

Debaryomyces hansenii is a yeast with considerable biotechnological potential as an osmotolerant, stress-tolerant oleaginous microbe. However, targeted genome modification tools are limited and require a strain with auxotrophic markers. Gene targeting by homologous recombination has been reported to be inefficient, but here we describe a set of reagents and a method that allows gene targeting at high efficiency in wild-type isolates.

Peroxisomal NAD(H) Homeostasis in the Yeast Depends on Two Redox Shuttles and the NAD Carrier, Pmp47.

is considered an unconventional yeast with a strong biotechnological potential, which can produce and store high amounts of lipids. However, relatively little is known about its lipid metabolism, and genetic tools for this yeast have been limited. The aim of this study was to explore the fatty acid β-oxidation pathway in .

Contribution of domain structure to the function of the yeast DEDD family exoribonuclease and RNase T functional homolog, Rex1.

The 3' exonucleolytic processing of stable RNAs is conserved throughout biology. Yeast strains lacking the exoribonuclease Rex1 are defective in the 3' processing of stable RNAs, including 5S rRNA and tRNA. The equivalent RNA processing steps in are carried out by RNase T.

The exosome-binding factors Rrp6 and Rrp47 form a composite surface for recruiting the Mtr4 helicase.

The exosome is a conserved multi-subunit ribonuclease complex that functions in 3' end processing, turnover and surveillance of nuclear and cytoplasmic RNAs. In the yeast nucleus, the 10-subunit core complex of the exosome (Exo-10) physically and functionally interacts with the Rrp6 exoribonuclease and its associated cofactor Rrp47, the helicase Mtr4 and Mpp6. Here, we show that binding of Mtr4 to Exo-10 in vitro is dependent upon both Rrp6 and Rrp47, whereas Mpp6 binds directly and independently of other cofactors.

Exosome substrate targeting: the long and short of it.

The exosome ribonuclease complex functions in both the limited trimming of the 3'-ends of nuclear substrates during RNA processing events and the complete destruction of nuclear and cytoplasmic RNAs. The two RNases of the eukaryotic exosome, Rrp44 (rRNA-processing protein 44) and Rrp6, are bound at either end of a catalytically inert cylindrical core. RNA substrates are threaded through the internal channel of the core to Rrp44 by RNA helicase components of the nuclear TRAMP complex (Trf4-Air2-Mtr4 polyadenylation complex) or the cytoplasmic Ski (superkiller) complex.

The exosome cofactor Rrp47 is critical for the stability and normal expression of its associated exoribonuclease Rrp6 in Saccharomyces cerevisiae.

Rrp6 is a conserved catalytic subunit of the eukaryotic nuclear exosome ribonuclease complex that functions in the productive 3' end maturation of stable RNAs, the degradation of transiently expressed noncoding transcripts and in discard pathways that eradicate the cell of incorrectly processed or assembled RNAs. The function of Rrp6 in these pathways is at least partially dependent upon its interaction with a small nuclear protein called Rrp47/Lrp1, but the underlying mechanism(s) by which Rrp47 functions in concert with Rrp6 are not established. Previous work on yeast grown in rich medium has suggested that Rrp6 expression is not markedly reduced in strains lacking Rrp47.

Rrp47 functions in RNA surveillance and stable RNA processing when divorced from the exoribonuclease and exosome-binding domains of Rrp6.

The eukaryotic exosome exoribonuclease Rrp6 forms a complex with Rrp47 that functions in nuclear RNA quality control mechanisms, the degradation of cryptic unstable transcripts (CUTs), and in the 3' end maturation of stable RNAs. Stable expression of Rrp47 is dependent upon its interaction with the N-terminal domain of Rrp6 (Rrp6NT). To address the function of Rrp47 independently of Rrp6, we developed a DECOID (decreased expression of complexes by overexpression of interacting domains) strategy to resolve the Rrp6/Rrp47 complex in vivo and employed mpp6Δ and rex1Δ mutants that are synthetic lethal with loss-of-function rrp47 mutants.

Assembly of the yeast exoribonuclease Rrp6 with its associated cofactor Rrp47 occurs in the nucleus and is critical for the controlled expression of Rrp47.

Rrp6 is a key catalytic subunit of the nuclear RNA exosome that plays a pivotal role in the processing, degradation, and quality control of a wide range of cellular RNAs. Here we report our findings on the assembly of the complex involving Rrp6 and its associated protein Rrp47, which is required for many Rrp6-mediated RNA processes. Recombinant Rrp47 is expressed as a non-globular homodimer.

Rrp6, rrp47 and cofactors of the nuclear exosome.

This chapter reviews the present state of knowledge on the activity of enzymes that function with the RNA exosome in the nucleus. In this compartment, the exosome interacts physically and functionally with the exoribonuclease Rrp6 and several cofactors, most prominently Rrp47 and the TRAMP complex. These interactions decide the fate of RNA precursors from transcription through the formation of mature ribonucleoprotein particles (RNPs) and the export of the RNPs to the cytoplasm.

Finding the exosome.

We describe the events surrounding the identification of the exosome complex and the subsequent early development of the field. Like many scientific discoveries, the initial identification and characterization of the exosome was based on a combination of skill, good fortune-and the availability of cutting edge technology.

The C-terminal region of the exosome-associated protein Rrp47 is specifically required for box C/D small nucleolar RNA 3'-maturation.

Cells lacking the exosome-associated protein Rrp47 show similar defects in stable RNA processing to those observed in the absence of the catalytic subunit Rrp6, but the precise mechanism(s) by which Rrp47 functions together with Rrp6 remains unclear. Deletion complementation analyses defined an N-terminal region of Rrp47, largely coincident with the bioinformatically defined Sas10/C1D domain, which was sufficient for protein function in vivo. In vitro protein interaction studies demonstrated that this domain of Rrp47 binds the PMC2NT domain of Rrp6.

Rrp47 and the function of the Sas10/C1D domain.

The Sas10/C1D domain is found in a small group of eukaryotic proteins that have functions in RNA processing events, translational control and DNA repair mechanisms. The domain is predicted to be alpha-helical in nature and comprises approx. 80 amino acid residues.

Rrp6, Rrp47 and cofactors of the nuclear exosome.

This chapter reviews the present state of knowledge on the activity of enzymes that function with the RNA exosome in the nucleus. In this compartment, the exosome interacts physically and functionally with the exoribonuclease Rrp6 and several cofactors, most prominently Rrp47 and the TRAMP complex. These interactions decide the fate of RNA precursors from transcription through the formation of mature ribonucleoprotein particles (RNPs) and the export of the RNPs to the cytoplasm.

Finding the exosome.

We describe the events surrounding the identification of the exosome complex and the subsequent early development of the field. Like many scientific discoveries, the initial identification and characterization ofthe exosome was based on a combination of skill, good fortune--and the availability of cutting edge technology.

The PMC2NT domain of the catalytic exosome subunit Rrp6p provides the interface for binding with its cofactor Rrp47p, a nucleic acid-binding protein.

The exosome complex is a key component of the cellular RNA surveillance machinery and is required for normal 3' end processing of many stable RNAs. Exosome activity requires additional factors such as the Ski or TRAMP complexes to activate the complex or facilitate substrate binding. Rrp47p promotes the catalytic activity of the exosome component Rrp6p, but its precise function is unknown.