A decrease in secretory protein mRNAs under endoplasmic
reticulum stress in filamentous fungi: Elucidation of themechanism and application in protein secretory production
Takahiro SHINTANI
Graduate School of Agricultural Science, Tohoku University
Research objectives
When eukaryotic cells are exposed to dithiothreitol (DTT) or tunicamycin, unfolded
proteins are accumulated in the endoplasmic reticulum (ER), thereby activating transcriptionof the ER chaperone genes. This phenomenon is called unfolded protein response (UPR). In
addition to the UPR, filamentous fungi undergo a downregulation of gene expression ofsecreted proteins, called as repression under secretion stress (RESS), when treated withDTT1). Although RESS might be caused by the transcriptional repression of secretory
protein genes depending on their promoter sequences, the detail remains unclear.
We have shown that the UPR was activated in Aspergillus oryzae under theconditions inducing the production of amylolytic enzymes and in A. oryzae by producing anaberrant secretory protein2,3). These ER stresses could cause negative feedback on protein
production through RESS. Therefore, we aimed to elucidate the RESS mechanism in A.
oryzae.
Methods
Detection of RESS
A. oryzae strains were grown in Czapek-Dox (CD) liquid medium containing 1%
fructose and 0.1% peptone at 30ºC for 24 h, and then transferred to the same medium
containing 1% maltose instead of fructose, to induce an α-amylase expression. DTT wasadded to the culture at a final concentration of 20 mM and the mycelia were harvested after
appropriate periods. Total RNAs were prepared from the mycelia and subjected to northernblot analyses. In order to express ireA under the control of thiamine-repressible nmtA
promoter, media containing 10 µM thiamine were used.
Results1) Involvement of a promoter-dependent transcription repression in the decrease of
α-amylase mRNA
The amount of amyA/B/C (α-amylase) mRNAs was dramatically decreased in thewild type strain in response to DTT treatment. Since a transcript of amyR, which encodes a

transcriptional activator for amyA/B/C expression, was gradually decreased in this condition,
it was suggested that the RESS of amyA/B/C could be due to the transcriptional inactivationof amyA/B/C associated with the downregulation of AmyR. However, the RESS of amyB
was observed even when amyB was expressed under the control of the promoter of enoA
(enolase gene), whose expression is not influenced by AmyR. These results suggested thatthe promoter-dependent transcriptional repression was not the leading cause of the RESS ofamyA/B/C.
2) The amyA/B/C mRNAs are degraded dependently on IreA under ER stress
Nascent polypeptides being synthesized in the cytoplasm are targeted to the ER
membrane as a part of the tertiary complex with a ribosome and an mRNA. Therefore, it wasassumed that the mRNA of a secretory protein being synthesized might be destabilized by
the stressed ER. Accordingly, we constructed the strain that expressed the AmyB containing
frame shift mutation in its ER targeting signal (AmyB-FS) and analyzed the amount of theamyB-FS mRNA under the ER stress condition. We found that the decrease in the amyBmRNA was evaded by this mutation, suggesting that the decrease in the amyB mRNA waslargely due to the ER targeting of the mRNA.
Next, we investigated whether this phenomenon was regulated by the UPR. Underthe ER stress condition, the mRNA of bZIP-type transcription factor HacA undergoes
unconventional splicing by the ER membrane-spanning kinase/endoribonuclease IreA toinduce the UPR4). Accordingly, we analyzed the RESS in the strain repressing ireA andfound that the decrease in the amyA/B/C mRNAs was evaded by ireA repression. Similarly,
the amyA/B/C mRNA decrease was very limited in the ∆ireA cells expressing the spliced
form of HacA. These results suggested that the RESS may not be a downstream of the UPR,
but directly or indirectly regulated by IreA. Therefore, we assumed that the amyA/B/C
mRNAs were degraded in an IreA-dependent manner when the cells were treated with DTT.
To test this hypothesis, we attempted to detect the degradation products of the amyA/B/C
mRNAs in the ER stress condition. If an mRNA is cleaved by an endoribonuclease, theresulting mRNA fragments are degraded by exoribonucleases. To detect such mRNAfragments, we analyzed the RESS in the strain deleted for ski2, which encodes a subunit of
the exoribonuclease complex, exosome. Northern blot analysis revealed that the amyA/B/CmRNA fragments appeared after the DTT treatment, suggesting that mRNA decay might beinvolved in the downregulation of the amyA/B/C mRNAs.
Conclusion
In this study, we showed that mRNA decay was involved in the downregulation ofgene expression of secretory proteins under ER stress elicited with DTT. We assume that themRNA targeted to the ER membrane as a complex with the nascent polypeptides andribosomes may undergo cleavage by IreA and the resulting mRNA fragments may bedegraded by exosomes. The decay of the mRNAs of secretory proteins under ER stress has
been reported in Drosophila melanogaster for the first time and named as “regulated
Ire1-dependent decay (RIDD)”5). RIDD was also found in mammals and plants. Our study
suggested that filamentous fungi also utilize the RIDD mechanism to downregulate gene
expression of secretory proteins. If the ER stress elicited by the overexpression of exogenous
secretory proteins activates RIDD, it could be a bottleneck in protein production withfilamentous fungi. It is necessary to analyze whether the overexpression of exogenous
secretory proteins induces RIDD in filamentous fungi.
Acknowledgment
This work was conducted in corporation with Jun-ichi Yokota, Yuko Sugiyama, ShunSato, Dr. Mizuki Tanaka, Dr. Silai Zhang, and Prof. Katsuya Gomi.

Figure A model of amyB mRNA decay induced by dithiothreitol treatment
Wild type amyB mRNA is recruited to the endoplasmic reticulum (ER) membrane
dependently on the ER targeting signal of nascent polypeptide and undergoes degradation.
The amyB-FS mRNA containing the frame shift mutation in its sequence coding the ERtargeting signal is unable to be targeted to the ER and thus evades its degradation.

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