Genetic And Molecular Biology Analysis Of Arabidopsis Mom1 Mutant

The transcription is tightly controlled in eukaryotic cells. mRNA shares common features, including coding region,5'and 3'-untranslated regions (UTRs). Most mRNA regulatory elements are located in the 5'and 3'UTRs. The 5'UTR mainly functions in regulating mRNA translation, whereas the 3'UTR plays important roles in nuclear export, cytoplasmic localization, translational efficiency, and mRNA stability. The 3'UTR sequence was recognized by polyA complex that promotes transcript cleavage at the polyA site and polyadenylation that in turn directs RNA polymeraseâ…¡-mediated termination. Recent studies indicated that transcriptional termination enhances pre-mRNA processing and protein expression, and aberrant or lacking 3'UTR leads to improperly terminated and unpolyadenylated mRNA which is degraded by RNA dependent RNA polymerase6 (RDR6)-mediated post-transcriptional gene silencing (PTGS). Several RNA surveillance pathways, such as nonsense-mediated mRNA decay (NMD), exist in cells for rapidly removing aberrant RNAs. However, it is not well understood how aberrant and/or readthrough mRNA is confined in the transcription initiation stage.To study the machinery responsible for preventing aberrant RNA transcriptional read-through, we constructed two chimeric genes:the coding sequence (CDS) of firefly LUC under the control of the CaMV 35SP in the presence (35SP-LUC-3'UTR) or absence (35SP-LUC) of a CaMV 35S 3'-UTR fragment, and both of them had a complete GUS gene drived by CaMV 35SP. In contrast to the LUC plants (harboring 35SP-LUC-3'UTR), of which 90% were LUC luminance-positive, no LUC was detected in more than 80% of the WT plants (harboring 35SP-LUC). The Arabidopsis mom1-44 mutation was identified by luciferase imaging a stable WT lines that no luminescent phenotype.Compared the GUS gene in WT plants, we could not detect any LUC transcript and protein in WT plants. Though both the LUC and GUS transcription level were up-regulated in moml-44 mutant, the LUC transcript was about 4kb more large than the expected 2kb in size and mostly polyadenylated, LUC protein was the normal size and functional. However, LUC transcript was mostly unpolyadenylated and unstable in WT plants. These results suggest that MOM1 restricts aberrant RNA transcription.Further studies had shown that, in WT plants, both of the CaMV 35SPs that drived LUC and GUS gene were heavily methylated. We could detect the small RNAs were generated from 35SP regions. In moml-44 mutant, the methylated on CaMV 35SP regions were same as WT, but small RNA was down-regulated. CaMV 35SP small RNA could not detect in rdr2 mutant, LUC gene was still silence; and the LUC gene in rdr2mom1-44 double mutant was same as in mom1-44 single mutant. These results indicated that MOM1 restricting aberrant RNA transcriptional read-through is independent of RdDM pathyway.In mom1-44 mutant, transcription read-though occurred in LUC gene with aberrant 3'UTR and that is associated with the H3K4 and H3K9 histone modification of their transcript regions. But GUS corresponding region was same as WT plants, and MOM1 restricts aberrant RNA transcription is independent of gene position copy numbers. Moreover, through analysis of MOM 1 functional domains, we determined that the CMM2 (Conserved MOM1 Motif2) is necessary to suppression of aberrant RNA transcription read-through.Overall, in this study, we provide evidence that MOM1 is required to prevent aberrant RNA transcriptional read-through in Arabidopsis.