SUMOylation Involves In Repression Of RNA Polymerase V (Pol V)-mediated Aberrant Gene Transcriptional Read-through In Arabidopsis

Three primer untranslated region (3’-UTR), a fraction of mRNA that immediately follows translation termination codon, is derived from 3’flanking region of one gene. Previous reports have shown that 3’-UTR participates in numerous regulatory processes including pre-mRNA cleavage, polyadenylation, stability and localization of mRNA and translation efficiency, indicating that 3’-UTR plays a vital role in accurate regulation of gene expression. RDR6-mediated RNA silencing mechanism can be induced when some genes missing 3’-UTR generate improperly terminated and unpolyadenylated mRNAs in Arabidopsis. This surveillance pathway contributes to the integrity of gene expression in a post-transcriptional regulation way; however, little is known how the expression of these aberrant genes is controlled at transcriptional level.Transcriptional readthrough (TRT) occurs when one gene passes the termination signal in the process of transcription. TRT can cause transcriptional interference at neighboring genes and produce aberrant readthrough transcript.3’-UTR participates in effective transcription termination, which helps to prevent TRT and transcriptional interference at neighboring genes. Although previous research has extensively studied transcription termination, the regulation mechanism underlying transcription termination of genes lacking 3’-UTR remains poorly understood.To explore the mechanism of restricting TRT of genes lacking 3’-UTR at transcriptional level, we constructed a plasmid carrying a LUCIFERASE (LUC) and a downstream hygromycin phosphotransferase (HPT) all driven by CaMV 35S, of which LUC lacks the 3’-UTR (35S-LUC). We transformed this plasmid into Col-0 ecotype and picked out a transgenic line, which was no LUC luminance, referred as wild type (WT). Then we mutagenized WT with ethyl methanesulfonate (EMS) and obtained a 438-1 mutant, which displayed a weak LUC luminescence. The mutant 438-1 rescued LUC activity because of TRT of LUC using 3’-UTR of the downstream gene HPT. Similarly, TRT also occurs at an endogenous mutator-like element MULE-F19G14 lacking 3’-UTR because of using 3’-UTR of the downstream gene CYP40. By map-based cloning and complementation assay, we identified that the mutation of OTS1 gene resulted in TRT in mutant 438-1, hence we renamed 438-1 to ots1-3. It’s known that OTS1 (OVERLY TOLERANT TO SALT1) is a SUMO protease, whose deSUMOylation activity involves in many physiological processes in plants, such as salt stress response and flowering regulation. Here we found that SUMO protease activity of OTS1 is also required for inhibiting TRT of LUC and MULE-F19G14. Our previous experiments showed that MOM1 also restricts TRT of these aberrant genes. Here, the results implied that OTS1 and MOM1 may function in different pathways to mediate this process. Genetic results showed that mutations of Pol V or DDR complex inhibited TRT of LUC and MULE-F19G14 in otsl-3, indicating that TRT depend on Pol V and DDR complex in ots1-3. Furthermore, we found that the largest subunit of Pol V, NRPE1, which interacts with OTS1 and SIZ1, can be modified by SUMO1 in vivo, implying that OTS1 and SIZ1 may mediate SUMO modification of Pol V. Finally, reverse genetics showed that other known and putative SUMO proteases except OTS2 and ULP21ike2 and two SUMO E3 ligases have similar effects on silencing for these aberrant genes, reflecting that SUMO modification plays an important role in transcriptional gene silencing (TGS) of genes lacking 3’-UTR.Taken together, our results reveal a Pol V-involved and SUMOylation-dependent regulatory mechanism for restricting TRT of aberrant genes.