Recombinant Debaryomyces hansenii RNA exonuclease 4 (REX4)

Function and Role of REX4

REX4 is involved in the processing of the internal transcribed spacer 1 (ITS1) region of pre-rRNA, which is essential for the production of mature 5.8S rRNA . Specifically, REX4 influences the ratio of short (5.8S S) to long (5.8S L) forms of 5.8S rRNA .

• REX4's role in rRNA processing was discovered through genetic studies involving mutants with temperature-sensitive mutations affecting RNase MRP, an enzyme responsible for cleavage at site A3 within the ITS1 region .

• Inactivation of the REX4 gene in these mutants restores cleavage at site A3 and shifts the production of 5.8S rRNA back to the short form, indicating REX4's involvement in ITS1 processing .

• Further analysis revealed that in certain mutant cells, ITS1 is processed at an alternative site (A4) in a REX4-dependent manner .

Genetic Interactions and Effects of REX4 Inactivation

Inactivation of the REX4 gene can reverse the effects of other mutations affecting pre-rRNA processing . For example, in rrp2-1 mutant cells, which exhibit aberrant pre-rRNA processing, inactivation of REX4 negates the effects of the original mutation .

• In rrp2-1 mutants, the order of ITS1 and ITS2 processing is reversed, but this phenotype disappears upon REX4 inactivation .

• Inactivation of REX4 in rrp2-1 mutants also restores the steady-state level of 7S pre-rRNA, which is reduced in the single mutant .

REX4 interacts genetically with other RNA catabolic enzymes, such as Rnt1p and Dbr1p, linking these enzymes to ITS1 processing and 5.8S rRNA production .

Debaryomyces Hansenii and its Biotechnological Significance

Debaryomyces hansenii exhibits several characteristics that make it valuable for various biotechnological applications :

• Stress Tolerance: D. hansenii can grow under extreme conditions, including high salt concentrations and alkaline pH levels, making it suitable for biopreservation in high-salt environments .

• Metabolic Versatility: It can utilize different carbon sources and produce various enzymes and metabolites, such as xylitol, lipases, and β-glucosidases, which are useful in the food and fuel industries .

• Genetic Manipulation: CRISPR-Cas9 tools have been developed for efficient genome editing in D. hansenii, allowing for precise gene inactivation and modification .

D. hansenii has the highest coding capacity among yeasts, making it a valuable eukaryotic microorganism model for studying osmotic adaptations and salt tolerance . It can also improve plant nutrition and stimulate growth, showing potential as a biofertilizer .

Experimental Evidence of REX4 Function

Experiments using primer extension analysis have provided direct evidence for REX4's role in ITS1 processing .

• Primer extension analysis of RNA isolated from rrp2-1 mutant cells shows a clear stop at site A4, indicating ITS1 processing at this alternative site .

• Inactivation of REX4 in the rrp2-1 mutant eliminates the signal at site A4, confirming that this processing is REX4-dependent .

These findings demonstrate that REX4 is required for the altered ITS1 processing observed in rrp2-1 mutant cells .

Data Table: Effects of REX4 Inactivation on Pre-rRNA Processing in rrp2-1 Mutant Cells