MORPHEUS' MOLECULE1 is required to prevent aberrant RNA transcriptional read-through in Arabidopsis

Abstract

Several pathways function to remove aberrant mRNA in eukaryotic cells; however, the exact mechanisms underlying the restriction of aberrant mRNA transcription are poorly understood. In this study, we found that MORPHEUS' MOLECULE1 (MOM1) is a key component of this regulatory machinery. The Arabidopsis (Arabidopsis thaliana) mom1-44 mutation was identified by luciferase imaging in transgenic plants harboring a cauliflower mosaic virus 35S promoter-LUCIFERASE transgene lacking the 3'-untranslated region. In the mom1-44 mutant, transcriptional read-though occurred in genes with an aberrant RNA structure. Analysis of an RNA-dependent RNA polymerase2 mom1 double mutant revealed that the RNA-directed DNA methylation pathway is not involved in this regulatory process. Moreover, the prevention of aberrant mRNA transcriptional read-through by MOM1 is gene locus and transgene copy number independent.

Figure 1.

Identification of the M-44 mutant. A, Schematic diagram of the LUC constructs in the binary vector pCAMBIA1301. Both T-DNA regions contain the complete GUS and HPT genes driven by the CaMV 35SP and the 35SP-controlled LUC CDS with (35SP-LUC-3′UTR) or without (35SP-LUC) the 3′-UTR. E35S, CaMV 35SP; 3′-UTR, 35S terminator; LB, left border; RB, right border. B, GUS staining of a wild-type (WT) seedling. C, Luminescence images showing LUC expression in M-44 mutant, wild-type (35SP-LUC), and LUC (35SP-LUC-3′UTR) transgenic plants. D, Luminescence intensity indicating LUC expression in C. One representative experiment of three replicates is shown. Error bars represent sd (n = 20).

Figure 2.

Positional cloning and confirmation of MOM1. A, Genetic mapping delimited M-44 to bacterial artificial chromosome clone (BAC) T6D22. B, Structure of MOM1 and position of the M-44/mom1-44 mutation. Boxes, exons; lines, introns. The mom1-44 mutation and T-DNA insertions in mom1-2 and mom1-3 are indicated by arrows. The M-44 mutation is located 400 bp downstream of the MOM1 transcription start site. C, Expression of MOM1 in Col-0, mom1-2, and mom1-3 as shown by RT-PCR. ACTIN was used as a control. D, M-44/mom1-44 is allelic to mom1. M-44/mom1-44 was crossed to wild-type (WT), mom1-2, or mom1-3 plants. LUC imaging was done using 7-d-old F1 wild-type or mom1 mutant seedlings. E, Complementation of mom1-44 as shown by LUC imaging. [See online article for color version of this figure.]

Figure 3.

LUC and GUS expression in wild-type (WT) and mom1-44 plants. A, Nuclear run-on assays of LUC and GUS. ACTIN was used as a control. B, Transcript levels and sizes of LUC and GUS. ACTIN was used as a control. C, Protein expression of LUC and GUS in wild-type and mom1-44 seedlings. Rubisco was used as a control.

Figure 4.

LUC transcript status in wild-type (WT) and mom1-44 plants. A, Expression analysis of LUC and HPT in LUC, wild-type, and mom1-44 plants by northern blotting using poly(A)⁺ (right) or total (left) RNA. B, Expression analysis of LUC in wild-type and mom1-44 plants by RT-PCR using total RNA reverse transcribed using oligo(dT) (left) or random (right) primers. Primers used were F0 and R0. ACTIN was used as a loading control. C, Relative LUC expression in wild-type plants as shown by real-time PCR using total RNA reverse transcribed using oligo(dT) (left) or random (right) primers. Primers used were F0 and R0. ACTIN was used as an internal control. D, Structure of the 35SP-LUC and 35SP-HPT-3′UTR clusters. The positions of the primers used are indicated by arrows. Two forms of LUC mRNA were observed in the mom1-44 mutant (the positions are indicated by arrows): an unpolyadenylated transcript located 130 bp downstream of the LUC translation stop codon and a polyadenylated transcript produced using the HPT 3′-UTR located 2,304 bp downstream of the LUC translation stop codon. E, LUC transcriptional read-through was detected in mom1-44. The 4-kb LUC transcript was polyadenylated and terminated using the HPT 3′-UTR. The cDNAs were reverse transcribed from LUC plants (L), wild-type plants (W), and mom1-44 (M) plants. D indicates genomic DNA from mom1-44.

Figure 5.

Endogens MULE-F19G14 transcript status in wild-type (WT) and mom1-44 plants. A, Scheme of the chromosomal region containing MULE-F19G14 and CYP40. The gray box represents MULE-F19G14; dark and open boxes represent exons and introns of CYP40, respectively. B, A 7-kb-long transcript of MULE-F19G14 was only detected in mom1-44 by northern-blotting assay. rRNA was used as a loading control. C, Relative MULE-F19G14 expression in wild-type plants as shown by real-time PCR using total RNA reverse transcribed using oligo(dT) (left) or random (right) primers. ACTIN was used as an internal control. D, Expression analysis of MULE-F19G14 in wild-type and mom1-44 plants by RT-PCR using total RNA reverse transcribed using oligo(dT) (left) or random (right) primers.

Figure 6.

35SP-directed siRNA production in wild-type (WT) and mom1-44 plants is not related to LUC transcriptional read-through. A, Detection of CaMV 35SP-directed siRNA, miR166, and siRNA02 in the indicated genotypes. An ethidium bromide-stained gel corresponding to tRNA and 5S rRNA was used as a loading control. The positions of the size markers are indicated (24 or 21 nucleotides [nt]). B, LUC luminescence in mom1-44, rdr2, and rdr2 mom1-44 mutant plants. The plants shown (from top to bottom) are as follows: wild type, mom1-44, rdr2, and rdr2 mom1-44. C, Expression of LUC in LUC, wild-type, mom1-44, rdr2, and rdr2 mom1-44 plants. Ethidium bromide-stained rRNA was used as a loading control. [See online article for color version of this figure.]

Figure 7.

LUC transcriptional read-through in mom1 is independent of the insertion position, copy number, and RDR2. A, Luminescence intensity (LUC expression) in various genetic backgrounds. Error bars represent sd (n = 20). B, Expression of 4-kb LUC and GUS transcripts in LUC-negative 35S-LUC transgenic seedlings (six independent lines) produced in the Col-0 background. rRNA was used as a loading control.