1. Comprehensive structural annotation of Pichia pastoris transcriptome and the response to various carbon sources using deep paired-end RNA sequencing
- Author
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Ying Lin, Shuli Liang, Xiaoning Wang, Bin Wang, Shuangyan Han, Yanrui Ye, Suiping Zheng, Minghui He, and Li Pan
- Subjects
Untranslated region ,lcsh:QH426-470 ,lcsh:Biotechnology ,Molecular Sequence Data ,RNA-Seq ,Bioengineering ,Computational biology ,Translation initiation mechanism ,Pichia ,Pichia pastoris ,Transcriptome ,Methanol induction ,lcsh:TP248.13-248.65 ,Genetics ,Gene ,biology ,Sequence Analysis, RNA ,Gene Expression Profiling ,Alternative splicing ,Intron ,High-Throughput Nucleotide Sequencing ,Molecular Sequence Annotation ,biology.organism_classification ,Blotting, Northern ,Carbon ,Introns ,Internal ribosome entry site (IRES) ,Alternative Splicing ,lcsh:Genetics ,MRNA Sequencing ,Genome, Fungal ,Research Article ,Biotechnology - Abstract
Background The methylotrophic yeast Pichia pastoris is widely used as a bioengineering platform for producing industrial and biopharmaceutical proteins, studying protein expression and secretion mechanisms, and analyzing metabolite synthesis and peroxisome biogenesis. With the development of DNA microarray and mRNA sequence technology, the P. pastoris transcriptome has become a research hotspot due to its powerful capability to identify the transcript structures and gain insights into the transcriptional regulation model of cells under protein production conditions. The study of the P. pastoris transcriptome helps to annotate the P. pastoris transcript structures and provide useful information for further improvement of the production of recombinant proteins. Results We used a massively parallel mRNA sequencing platform (RNA-Seq), based on next-generation sequencing technology, to map and quantify the dynamic transcriptome of P. pastoris at the genome scale under growth conditions with glycerol and methanol as substrates. The results describe the transcription landscape at the whole-genome level and provide annotated transcript structures, including untranslated regions (UTRs), alternative splicing (AS) events, novel transcripts, new exons, alternative upstream initiation codons (uATGs), and upstream open reading frames (uORFs). Internal ribosome entry sites (IRESes) were first identified within the UTRs of genes from P. pastoris, encoding kinases and the proteins involved in the control of growth. We also provide a transcriptional regulation model for P. pastoris grown on different carbon sources. Conclusions We suggest that the IRES-dependent translation initiation mechanism also exists in P. pastoris. Retained introns (RIs) are determined as the main AS event and are produced predominantly by an intron definition (ID) mechanism. Our results describe the metabolic characteristics of P. pastoris with heterologous protein production under methanol induction and provide rich information for further in-depth studies of P. pastoris protein expression and secretion mechanisms.
- Published
- 2012