Bicistronic and fused monocistronic transcripts are derived from adjacent loci in the Arabidopsis genome

Abstract

Comparisons of full-length cDNAs and genomic DNAs available for Arabidopsis thaliana described here indicate that some adjacent loci are transcribed into extremely long RNAs spanning two annotated genes. Once expressed, some of these transcripts are post-transcriptionally spliced within their coding and intergenic sequences to generate bicistronic transcripts containing two complete open reading frames. Others are spliced to generate monocistronic transcripts coding for fusion proteins with sequences derived from both loci. RT-PCR of several P450 transcripts in this collection indicates that these extended transcripts exist side by side with shorter monocistronic transcripts derived from the individual loci in each pair. The existence of these unusual transcripts highlights variations in the processes of transcription and splicing that could not possibly have been predicted in the algorithms used for genome annotation and splice site predictions.

FIGURE 1.

CYP71B35 and CYP71B34 transcripts. The gene models for these loci are designated with the predicted start and stop codons for each open reading frame above the current TAIR gene models (top line) and termination codons in-frame with the P450 open reading frame shown with asterisks. GenBank accession numbers for ESTs and full-length cDNAs are shown below each diagram. REFSEQ NM_113537 is provisional.

FIGURE 2.

CYP705A15 and CYP705A16 transcripts. The gene models for these loci are designated with the predicted start and stop codons for each open reading frame above the current TAIR gene models (top line) and termination codons in-frame with the P450 open reading frame designated with asterisks. GenBank accession numbers for ESTs and full-length cDNAs are shown below each diagram.

FIGURE 3.

CYP97C1 and OMT transcripts. The gene models for these loci are designated with the predicted start and stop codons for each open reading frame above the current TAIR gene model (top line). GenBank accession numbers for ESTs and full-length cDNAs are shown below each diagram.

FIGURE 4.

CYP71B10 and At5g57250 transcripts. The gene models for these loci are designated with the predicted start and stop codons for each open reading frame above the current TAIR gene model (top line). GenBank accession numbers for ESTs and full-length cDNAs are shown below each diagram. REFSEQ NM_125108 is provisional.

FIGURE 5.

CYP96A9 and CYP96A10 transcripts. The gene models for these loci are designated with the predicted start and stop codons for each open reading frame above the current TAIR gene model (top line). GenBank accession numbers for ESTs and full-length cDNAs are shown below each diagram. REFSEQ NM_120108

FIGURE 6.

CYP71A27 and CYP71A28 transcripts. The gene models for these loci are designated with the predicted start and stop codons for each open reading frame above the previous TAIR gene model (top line). One nucleotide addition and two nucleotide deletions introduced to generate the gene model available at http://www.biobase.dk/P450/Arab_cyps/(middle line) are designated with +1 and −2, respectively. The single EST available for this locus is shown below the diagram. REFSEQ NM_118143 is provisional.

FIGURE 7.

RT-PCR analysis. Total RNAs isolated from the aboveground tissues of unstressed 1-mo-old plants (control), cold and drought stressed 7-d-old seedlings (cold + drought), unstressed 7-d-old seedling shoots or roots, 3-wk-old rosettes, and 1-mo-old leaves, stems, or flowers were RT-PCR amplified in one-step or two-step reactions as outlined in Materials and Methods. The RT-PCR products were electrophoresed on 1.0% agarose gels, blotted to Hybond-N nylon membranes, and probed with ³²P-labeled gene-specific fragments.

FIGURE 8.

Classes of bicistronic and monocistronic transcripts derived from adjacent loci. Diagrams for two adjacent loci are depicted with their intergenic region (IGR). White boxes indicate that each ORF is as annotated in current gene models for the individual loci with all introns spliced as predicted. Gray boxes indicate that the ORF is altered from predictions due to splicing of introns at alternate positions within the coding region of the transcript, to N-terminal extension of coding sequences due to transcript initiation at sites upstream from the predicted translation start site, or to N-terminal truncation of coding sequences due to placement of the 5′ end of the transcript downstream from the predicted translation start site. Vertical lines designate the translation start and stop codons used in each ORF. The numbers of cDNAs in each category are as follows: A (7), B (2), C–J (1 each), K (2), L–N (1 each), O (7), P (12), Q (6), R (1), S (5), T (1), U (1), V (3), W (2).

FIGURE 9.

Intron junctions in intergenic regions of bicistronic transcripts.