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. 2012 Dec 31:13:738.
doi: 10.1186/1471-2164-13-738.

Comprehensive structural annotation of Pichia pastoris transcriptome and the response to various carbon sources using deep paired-end RNA sequencing

Shuli Liang  1 , Bin Wang, Li Pan, Yanrui Ye, Minghui He, Shuangyan Han, Suiping Zheng, Xiaoning Wang, Ying Lin
Affiliations

Comprehensive structural annotation of Pichia pastoris transcriptome and the response to various carbon sources using deep paired-end RNA sequencing

Shuli Liang et al. BMC Genomics. .

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.

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Figures

Figure 1
Figure 1
Mapping summary of RNA-Seq reads and transcriptional distribution in P. pastoris genome. (A) Statistics of RNA-Seq reads mapped in P. pastoris genome. Uniquely mapped reads include the reads located in exons, introns and intergenic regions. (B) Box-and-whisker plots of log2-transformed RPKM for the following regions: intergenic regions, introns, exons and novel transcripts. Diamonds represent data outside of the 5th or 95th percentile. (C) Expression profile denoted by log2-transformed reads count, depicting the transcriptionally active region (TAR) in the P. pastoris genome. “Coverage” depicts the percentage of the P. pastoris genome covered by RNA-Seq reads, and “Gene” depicts the number of BOGAS P. pastoris genes in a window size of 6 kilobases. The chromosome numbers are shown on the left.
Figure 2
Figure 2
Transcript structure annotation of P. pastoris by RNA-Seq data. (A) UTR redefinition of the gene PAS_chr1-4_0666. (B) Length distribution of 5’ and 3’ UTRs of all annotated P. pastoris genes. (C) Annotated uORF in the 5’ UTR of gene PAS_chr1-3_0005. (D) Annotated uATG in the 5’ UTR of gene PAS_chr2-2_0282.
Figure 3
Figure 3
IRES activity identification of 5’UTRs of KOG1 and GCN2 mRNA detected by RNA-Seq. (A) Construction of vectors. The bicistronic vector pRml-LacZ containing the RML gene and the LacZ gene used as the negative control. Candidate IRESes were inserted between the RML gene and the LacZ gene, generating the pRml-UTR-LacZ. The expression of LacZ ORF in another control plasmid pLacZ was controlled by the GAP promoter. The arrows represent the primers used in PCR identification. (B) Detection of RML and β-galactosidase on MDT plate and MDX plate, respectively. (C) Identification of the integrity of bicistronic mRNAs with primer P1/P2. (D) Northern blot analysis of the transcripts from total RNA isolated from P. pastoris X33/pRml-LacZ, X33/pRml-GCN2-LacZ, X33/pRml-KOG1-LacZ, and X33/pLacZ.
Figure 4
Figure 4
Novel transcripts and new exons detected in the P. pastoris transciptome by RNA-Seq data. (A) A novel transcript with considerable expression level in contig FN392322 (GI number: 238033210). (B) Length distribution of all detected novel transcripts. (C) A new exon detected in gene PAS_chr4_0246.
Figure 5
Figure 5
AS events in P. pastoris. (A) Examples of the main AS types in P. pastoris, including retained intron (RI), alternative 5’ splice site (A5SS) and alternative 3’ splice site (A3SS). The ordinate “Expression” represents the coverage depth of each genome location. (B) Statistics of AS events in P. pastoris.
Figure 6
Figure 6
Analysis of P. pastoris differential gene expression between SDEM and SDEG culture. (A) Differential gene expression between P. pastoris SDEM and SDEG culture, analyzed by the ‘R’ (DESeq) software. (B) Metabolic pathway analysis of differentially expressed genes between SDEM and SDEG culture. Genes denoted by red font are up-regulated in SDEM condition compared to SDEG condition. Genes denoted by green font are down-regulated.
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References

    1. Daly R, Hearn MTW. Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. J Mol Recognit. 2005;18(2):119–138. doi: 10.1002/jmr.687. - DOI - PubMed
    1. Hamilton SR, Gerngross TU. Glycosylation engineering in yeast: the advent of fully humanized yeast. Curr Opin Biotechnol. 2007;18(5):387–392. doi: 10.1016/j.copbio.2007.09.001. - DOI - PubMed
    1. Hamilton SR, Davidson RC, Sethuraman N, Nett JH, Jiang Y, Rios S, Bobrowicz P, Stadheim TA, Li H, Choi BK. et al.Humanization of yeast to produce complex terminally sialylated glycoproteins. Science. 2006;313(5792):1441–1443. doi: 10.1126/science.1130256. - DOI - PubMed
    1. Jacobs PP, Geysens S, Vervecken W, Contreras R, Callewaert N. Engineering complex-type N-glycosylation in Pichia pastoris using GlycoSwitch technology. Nat Protoc. 2009;4(1):58–70. - PubMed
    1. Gasser B, Maurer M, Rautio J, Sauer M, Bhattacharyya A, Saloheimo M, Penttilä M, Mattanovich D. Monitoring of transcriptional regulation in Pichia pastoris under protein production conditions. BMC Genomics. 2007;8(1):179. doi: 10.1186/1471-2164-8-179. - DOI - PMC - PubMed

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