Second-Site Mutagenesis of a Hypomorphic argonaute1 Allele Identifies SUPERKILLER3 as an Endogenous Suppressor of Transgene Posttranscriptional Gene Silencing.
Second-site mutagenesis was performed on the argonaute1-33 (ago1-33) hypomorphic mutant, which exhibits reduced sense transgene posttranscriptional gene silencing (S-PTGS). Mutations in FIERY1, a positive regulator of the cytoplasmic 5'-to-3' EXORIBONUCLEASE4 (XRN4), and in SUPERKILLER3 (SKI3), a member of the SKI complex that threads RNAs directly to the 3'-to-5' exoribonuclease of the cytoplasmic exosome, compensated AGO1 partial deficiency and restored S-PTGS with 100% efficiency. Moreover, xrn4 and ski3 single mutations provoked the entry of nonsilenced transgenes into S-PTGS and enhanced S-PTGS on partially silenced transgenes, indicating that cytoplasmic 5'-to-3' and 3'-to-5' RNA degradation generally counteract S-PTGS, likely by reducing the amount of transgene aberrant RNAs that are used by the S-PTGS pathway to build up small interfering RNAs that guide transgene RNA cleavage by AGO1. Constructs generating improperly terminated transgene messenger RNAs (mRNAs) were not more sensitive to ski3 or xrn4 than regular constructs, suggesting that improperly terminated transgene mRNAs not only are degraded from both the 3' end but also from the 5' end, likely after decapping. The facts that impairment of either 5'-to-3' or 3'-to-5' RNA degradation is sufficient to provoke the entry of transgene RNA into the S-PTGS pathway, whereas simultaneous impairment of both pathways is necessary to provoke the entry of endogenous mRNA into the S-PTGS pathway, suggest poor RNA quality upon the transcription of transgenes integrated at random genomic locations.
Institut Jean-Pierre Bourgin, Unité Mixte de Recherche 1318, Institut National de la Recherche Agronomique (INRA), 78000 Versailles, France (A.Y., B.S., N.B., E.E.-M., G.L., J.-S.P., J.-B.M., T.E., H.V.); andDepartment of Molecular Biology, Max Planck Institute for Developmental Biology, D-72076 Tuebingen, Germany (J.C.).
Agnès
http://orcid.org/0000-0002-3811-6485
A
Yu
Institut Jean-Pierre Bourgin, Unité Mixte de Recherche 1318, Institut National de la Recherche Agronomique (INRA), 78000 Versailles, France (A.Y., B.S., N.B., E.E.-M., G.L., J.-S.P., J.-B.M., T.E., H.V.); andDepartment of Molecular Biology, Max Planck Institute for Developmental Biology, D-72076 Tuebingen, Germany (J.C.).
Baptiste
B
Saudemont
Institut Jean-Pierre Bourgin, Unité Mixte de Recherche 1318, Institut National de la Recherche Agronomique (INRA), 78000 Versailles, France (A.Y., B.S., N.B., E.E.-M., G.L., J.-S.P., J.-B.M., T.E., H.V.); andDepartment of Molecular Biology, Max Planck Institute for Developmental Biology, D-72076 Tuebingen, Germany (J.C.).
Nathalie
N
Bouteiller
Institut Jean-Pierre Bourgin, Unité Mixte de Recherche 1318, Institut National de la Recherche Agronomique (INRA), 78000 Versailles, France (A.Y., B.S., N.B., E.E.-M., G.L., J.-S.P., J.-B.M., T.E., H.V.); andDepartment of Molecular Biology, Max Planck Institute for Developmental Biology, D-72076 Tuebingen, Germany (J.C.).
Emilie
http://orcid.org/0000-0002-4017-6436
E
Elvira-Matelot
Institut Jean-Pierre Bourgin, Unité Mixte de Recherche 1318, Institut National de la Recherche Agronomique (INRA), 78000 Versailles, France (A.Y., B.S., N.B., E.E.-M., G.L., J.-S.P., J.-B.M., T.E., H.V.); andDepartment of Molecular Biology, Max Planck Institute for Developmental Biology, D-72076 Tuebingen, Germany (J.C.).
Gersende
http://orcid.org/0000-0002-4609-7587
G
Lepère
Institut Jean-Pierre Bourgin, Unité Mixte de Recherche 1318, Institut National de la Recherche Agronomique (INRA), 78000 Versailles, France (A.Y., B.S., N.B., E.E.-M., G.L., J.-S.P., J.-B.M., T.E., H.V.); andDepartment of Molecular Biology, Max Planck Institute for Developmental Biology, D-72076 Tuebingen, Germany (J.C.).
Jean-Sébastien
http://orcid.org/0000-0001-7859-7415
JS
Parent
Institut Jean-Pierre Bourgin, Unité Mixte de Recherche 1318, Institut National de la Recherche Agronomique (INRA), 78000 Versailles, France (A.Y., B.S., N.B., E.E.-M., G.L., J.-S.P., J.-B.M., T.E., H.V.); andDepartment of Molecular Biology, Max Planck Institute for Developmental Biology, D-72076 Tuebingen, Germany (J.C.).
Jean-Benoit
JB
Morel
Institut Jean-Pierre Bourgin, Unité Mixte de Recherche 1318, Institut National de la Recherche Agronomique (INRA), 78000 Versailles, France (A.Y., B.S., N.B., E.E.-M., G.L., J.-S.P., J.-B.M., T.E., H.V.); andDepartment of Molecular Biology, Max Planck Institute for Developmental Biology, D-72076 Tuebingen, Germany (J.C.).
Jun
J
Cao
Institut Jean-Pierre Bourgin, Unité Mixte de Recherche 1318, Institut National de la Recherche Agronomique (INRA), 78000 Versailles, France (A.Y., B.S., N.B., E.E.-M., G.L., J.-S.P., J.-B.M., T.E., H.V.); andDepartment of Molecular Biology, Max Planck Institute for Developmental Biology, D-72076 Tuebingen, Germany (J.C.).
Taline
T
Elmayan
Institut Jean-Pierre Bourgin, Unité Mixte de Recherche 1318, Institut National de la Recherche Agronomique (INRA), 78000 Versailles, France (A.Y., B.S., N.B., E.E.-M., G.L., J.-S.P., J.-B.M., T.E., H.V.); andDepartment of Molecular Biology, Max Planck Institute for Developmental Biology, D-72076 Tuebingen, Germany (J.C.) herve.vaucheret@versailles.inra.fr.
Hervé
H
Vaucheret
D017360:Arabidopsis / Q000235:genetics* / Q000254:growth & development; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D060565:Argonaute Proteins / Q000235:genetics*; D003593:Cytoplasm / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D030821:Plants, Genetically Modified; D011061:Poly A / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D018749:RNA, Plant / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D019076:Transgenes
C117457:AGO1 protein, Arabidopsis; D029681:Arabidopsis Proteins; D060565:Argonaute Proteins; D018749:RNA, Plant; D011993:Recombinant Fusion Proteins; C000608437:SKI3 protein, Arabidopsis; D049452:Green Fluorescent Proteins; D011061:Poly A