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The Expression of Matryoshka Gene Encoding a Homologue of Kunitz Peptidase Inhibitor Is Regulated Both at the Level of Transcription and Translation

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Abstract

The gene for Kunitz peptidase inhibitor-like protein (KPILP) contains nested alternative open reading frame (aORF) that controls expression of the maternal mRNA. The content of NbKPILP mRNA in intact leaves of Nicotiana benthamiana plant is low but increases significantly upon extended dark exposure or when foreign nucleic acid is overexpressed in the cells. The NbKPILP gene promoter along with the expressed nested aORF are likely to play an important role in maintaining the levels of NbKPILP mRNA. To elucidate the role of NbKPILP promoter, we isolated a fragment of N. benthamiana chromosomal DNA upstream of the NbKPILP transcription start, sequenced it, and created constructs in which reporter E. coli uidA gene coding for β-D-glucuronidase (GUS) was placed under control of the NbKPILP promoter. By assessing the efficacy of uidA mRNA synthesis directed by the NbKPILP promoter and 35S promoter of the cauliflower mosaic virus in a transient expression system, we showed that the levels of GUS accumulation were comparable for both promoters. Prolonged incubation of the agroinjected plants in the darkness stimulated accumulation of the uidA mRNA directed by the NbKPILP promoter. Our experiments indicate that along with regulation at the transcriptional level, expression of NbKPILP mRNA can be affected by expression of the nested aORF controlled by the polypurine block (PPB) located upstream of its start codon, since introduction of mutations in the PPB resulted in significant accumulation of the NbKPILP mRNA. Nucleotide replacement in the aORF start codon led to the drastic increase in the amounts of NbKPILP mRNA and its protein product.

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Abbreviations

aORF:

nested alternative reading frame

GUS:

β-D-glucuronidase

KPI:

Kunitz peptidase inhibitor

(Nb)KPILP:

(Nicotiana benthamiana) Kunitz peptidase inhibitor-like protein

PPB:

polypurine block

proNbKPILP :

NbKPILP promoter

RT-qPCR:

quantitative reverse transcription polymerase chain reaction

35S:

cauliflower mosaic virus (CaMV) 35S RNA promoter

(s)ORF:

(short) ORF

sPEP:

peptide encoded by sORF

3′-UTR:

3′-untranslated region

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Authors and Affiliations

  1. Vavilov Institute of General Genetics, Russian Academy of Sciences, 119991, Moscow, Russia

    E. V. Sheshukova, T. V. Komarova, N. M. Ershova & Y. L. Dorokhov

  2. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119991, Moscow, Russia

    T. V. Komarova, N. M. Ershova & Y. L. Dorokhov

  3. Sechenov First Moscow State Medical University, 119991, Moscow, Russia

    A. M. Bronstein

Authors
  1. E. V. Sheshukova
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  2. T. V. Komarova
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  3. N. M. Ershova
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  4. A. M. Bronstein
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  5. Y. L. Dorokhov
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Corresponding author

Correspondence to Y. L. Dorokhov.

Additional information

Original Russian Text © E. V. Sheshukova, T. V. Komarova, N. M. Ershova, A. M. Bronstein, Y. L. Dorokhov, 2018, published in Biokhimiya, 2018, Vol. 83, No. 10, pp. 1562–1571. Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM18-120, September 17, 2018.

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Sheshukova, E.V., Komarova, T.V., Ershova, N.M. et al. The Expression of Matryoshka Gene Encoding a Homologue of Kunitz Peptidase Inhibitor Is Regulated Both at the Level of Transcription and Translation. Biochemistry Moscow 83, 1255–1262 (2018). https://doi.org/10.1134/S0006297918100103

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  • DOI: https://doi.org/10.1134/S0006297918100103

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