Polyadenylation of rRNA- and tRNA-based yeast transcripts cleaved by internal ribozyme activity

Area of Science:

• Molecular Biology
• RNA Processing
• Eukaryotic Gene Expression

Background:

• Polyadenylation is crucial for mRNA stability, transport, and translation in eukaryotes.
• Typically, only RNA polymerase II transcripts undergo polyadenylation at their 3' ends.
• Ribonucleic acid (RNA) polymerase I and III synthesize ribosomal RNA (rRNA) and transfer RNA (tRNA), respectively, which are not usually polyadenylated.

Purpose of the Study:

• To investigate whether non-canonical transcripts (rRNA and tRNA) can be polyadenylated in yeast.
• To determine if independent 3' end generation can enable polyadenylation of RNA polymerase I and III transcripts.

Main Methods:

• Engineering yeast genes for ribosomal DNA (rDNA) and tRNA to include a self-cleaving hammerhead ribozyme sequence.
• Analyzing the processing and polyadenylation status of the resulting transcripts in vivo.
• Utilizing hammerhead ribozyme activity to generate distinct 5' and 3' cleavage products.

Main Results:

• The hammerhead ribozyme efficiently cleaved both the engineered rDNA-based and tRNA transcripts.
• Two stable cleavage products were generated from each transcript.
• The 5' cleavage product of both transcripts was successfully polyadenylated.
• This demonstrates polyadenylation of transcripts typically synthesized by RNA polymerase I and III.

Conclusions:

• In yeast, polyadenylation is not strictly limited to RNA polymerase II transcripts.
• Independent generation of a transcript's 3' end, via ribozyme activity, can facilitate polyadenylation.
• This finding expands our understanding of RNA processing flexibility in eukaryotes.