Figure 1.
Post-transcriptional and co-transcriptional splicing.
A) Spliceosome assembly begins with the U1 and U2 snRNPs. On the post-transcriptional splicing pathway, polyadenylated pre-mRNA is spliced (step one) to produce polyadenylated lariat-exon2, from which mature mRNA results on completion of step two. B) Splicing can occur during transcript elongation on the co-transcriptional splicing pathway. The RNA polymerase Pol II (shown in green) transcribes Exon1, the Intron and Exon2 (colours of the RNA indicate the corresponding region of the DNA, and the red oval indicates the cap on the 5′ end of the RNA). Splicing can occur after the transcription of specific sequences that trigger the assembly and activation of the spliceosome on the RNA. C) The transcription and splicing pathway. A polymerase proceeds from the active promoter complex (APC) through 15 irreversible steps that represent the transcription of nucleotides in Exon 1 and the Intron up until the branchsite. From the branchsite () until the end of Exon 2, a transition can be made to the co-transcriptional splicing path
as the first step of splicing can occur. The characteristics of post-transcriptional splicing steps one and two (
and
), and co-transcriptional splicing step two (
) are determined by model comparison.
Figure 2.
The Ribo1 reporter and its response to doxycycline.
(A) Schematic of the promoter, intron and exons of Ribo1. The positions of the RT-qPCR primers are shown by the arrow C1. (B) Induction and splicing of Ribo1 measured by RT-qPCR. (C) Induction and step 1 splicing of 3′SSRibo1. Step two of splicing is blocked and therefore mRNA is not produced. (D) Induction of 5′SSRibo1. Step one of splicing is blocked and therefore neither lariat-exon2 nor mRNA is produced. Points indicate pre-mRNA (blue), lariat-exon2 (green) and mRNA (black) data. Error bars show the standard error of three biological replicates. Solid lines are model predictions.
Figure 3.
The 3′ cleavage and polyadenylation of Ribo1 measured by RT-qPCR.
(A) Schematic of the Ribo1 gene. The positions of the alternative cDNA primers for uncleaved and cleaved/polyadenylated transcripts are shown by the C2 and oligo (dT) arrows respectively. (B) Points indicate uncleaved pre-mRNA (U-pre-mRNA), polyadenylated pre-mRNA (P-pre-mRNA) and polyadenylated lariat-exon2 (P-lariat-exon2) data. (C) Points indicate 3′ uncleaved lariat-exon2 (U-lariat-exon2), uncleaved mRNA (U-mRNA) and polyadenylated mRNA (P-mRNA) data. Error bars show the standard error of three biological replicates. Solid lines are model predictions.
Table 1.
Comparison of pathways I-VIII using optimal parameters by AIC.
Figure 4.
A heat map of AIC scores for pre-mRNA, lariat-exon2 and mRNA.
A- indicates the AIC score for all species in Expt 1 (uncleaved and polyadenylated); U- AIC for uncleaved species in Expt 2; and P- AIC for polyadenylated species in Expt 2. The spectrum red to white reflects the lowest (best) to highest (worst) AIC score calculated for each species using the optimal parameter values for each pathway (rows I-VIII). Each column is normalised to have a minimum value of 0 by subtracting the minimum AIC score in the column from each entry.