Mechanism for mRNA maturation control in basidiomycetes
Yoichi HONDA
Graduate School of Agriculture, Kyoto University
Research aims
Basidiomycete fungi have been the target of interesting research for their wide application
including edible use as well as industrial utilization in the production of bioactive
compounds, lectins, drug intermediates, fermentation foods, pretreatment of lignocelluloses
for biomass refinery, and bioremediation of polluted environments. Recently, it has beenreported that basidiomycetes lack most of the carbohydrate-modifying system present inother eukaryotic organisms, and we are attempting to develop a biopharmaceuticalglycoprotein production system in the oyster mushroom, Pleurotus ostreatus, using
recombinant gene expression combined with in vitro glycoengineering techniques.
Most studies on recombinant protein production using eukaryotic microorganisms havebeen done in ascomycete fungi such as Saccharomyces cerevisiae and Aspergillus oryzae.
However, there is little compatibility between ascomycete and basidiomycete expression
vectors. Moreover, even when homologous promoter sequences are used to drive
codon-optimized coding sequences, the expression of heterologous proteins inbasidiomycetes is still challenging. Elucidating the fundamental mechanisms for gene
expression control in basidiomycete fungi is paramount for their utilization as an industrial
microorganism. The aim of this research is to demonstrate the unique properties of themRNA maturation process in basidiomycetes in order to develop a reliable system forheterologous protein expression in these fungi.
Methods
We have developed a protoplast transfection system in the basidiomycete that makes it
possible to precisely evaluate the activity of each gene expression control system using atransient reporter gene. It has been difficult to compare the expression of reporter constructs
in a stable transformation system because the introduced gene constructs are integrated withdifferent copy numbers at random sites in the chromosome of each transformant. Using theprotoplast transfection system, a basic promoter structure was determined for gpd and ß-tub
genes in Ceriporiopsis subvermispora (to be published). In the following sections of thisreport, we describe the experimental results for the characterization of each step in thebasidiomycete mRNA maturation process.
Results① Function of introns
Stable transformation experiments in Schizophyllum commune have demonstrated that anintron is essential for efficient gene expression in this organism 1). This has become anestablished theory in basidiomycetes. We tested the function of introns in recombinant gene
expression by analyzing the expression of a transient gene transfected into C. subvermispora.
The results showed that there is a negligible difference in the expression of reporter geneconstructs with or without an intron, indicating that an intron is therefore dispensable foreffective gene expression in this fungus.
② Evaluation of terminator sequences
Generally, mRNA processing starts

duringtranscription but it has not been described whether
transcription terminators have an important role inbasidiomycete gene expression. Functional differencesamong terminator sequences have been mostly ignoredin the design of expression vectors. We demonstrated
there is differential gene expression of reporter geneconstructs harboring different terminators, such as thegpd and ß-tub terminators, in C. subvermispora (Figure1). These results suggest that to construct more
powerful expression cassettes, the use of a strong

Figure 1. Transfection analysis of
HygR maker plasmid with different
terminator sequences.
No ter: no terminator sequence.

terminator is recommended.
③ Analysis of the poly(A) signal
In filamentous fungi, it was reported thatpoly(A) signals are less conserved than thosein mammalian species and a series of AT-richstretches can act as poly(A) signal inAspergillus oryzae2). If AT-rich sequences cansimilarly act as poly(A) in basidiomycetes,
they could represent an obstacle for theproduction of full-length mRNAs coding forheterologous proteins. To our knowledge,poly(A) signals have never been investigatedFigure 2. Mutational analysis of putative polyA
signals in the C. subvermispora gpd terminatorin basidiomycetes. We conducted 1) determination of poly(A) sites of a reporter construct instable C. subvermispora transformants and 2) mutational analysis of estimated poly(A) sites
using a transfection system in C. subvermispora (Figure 2). Our results indicate that awell-conserved consensus sequence has a major function in polyadenylation and somecomplementary sequences with weak activity can also direct poly(A) addition to the 3’ endof mRNAs.
Conclusions
Through transient reporter gene expression using a transfection system, we demonstrated:that an intron is not essential for efficient gene expression, arguing against an establishedtheory. We also demonstrated that a transcriptional terminator can greatly influence gene
expression levels and that, besides a strong poly(A) signal that is conserved among various
eukaryotes, there is (an)other sequence(s) with weak polyadenylation activity in the selective
lignin degrading fungus, C. subvermispora.
These results may foster the industrial utilization of basidiomycetes through the stable andefficient production of various recombinant proteins in the future. It should be noted that thebest poster prize was awarded to the presentation of part of this research at the European
Conference on Fungal Genetics, which was held in Haifa, Israel, in February 2018.
References
1) Lugones L.G., Scholtmeijer K., Klootwijk R., and Wessels J.G. (1999) Introns arenecessary for mRNA accumulation in Schizophyllum commune. Mol Microbiol. 32:
681-689.
2) Tanaka M., Sakai Y., Yamada O., Shintani T., and Gomi K. (2011) In silico analysis of3'-end-processing signals in Aspergillus oryzae using expressed sequence tags andgenomic sequencing data. DNA Res.18,189-200.
3) Honda Y., Nguyen D.X., Nishisaka E., Sakaguchi T., Nakazawa T., and Sakamoto M.
(2018) Functional analysis of gene expression signals in basidiomycetous fungi usingtransfection, and random or targeted integration on the chromosome.: EuropeanConference on Fungal Genetics, Haifa, Israel