1. Volume 18, Issue 2, Pages 245-251 (April 2005)
Delay in Synthesis of the 3′ Splice Site Promotes trans-Splicing of the Preceding 5′ Splice Site 
Terunao Takahara, Bosiljka Tasic, Tom Maniatis, Hiroshi Akanuma, Shuichi Yanagisawa 
Molecular Cell 
Volume 18, Issue 2, Pages (April 2005)
DOI: /j.molcel
Copyright © 2005 Elsevier Inc. Terms and Conditions

2. Figure 1
Shortened Third Intron Leads to Abolition of trans-Splicing between Sp1 pre-mRNAs
(A) Schematic representation of the endogenous Sp1 gene and integrated transgenes (C1, C kb int, C kb int, C2, C kb int, and C kb int). The Sp1 gene promoter was located immediately upstream of exon 1. Unique restriction enzyme sites (EcoRI and XhoI) were created in the transgenes. Exons are shown as boxes. The polyadenylation signal (pA) is from bovine growth hormone gene (Goodwin and Rottman, 1992).
(B) Expected structures of RT-PCR products derived from the endogenous Sp1 gene and the integrated transgenes. The origins of RT-PCR products are identified by restriction enzyme digestion. The expected sizes of RT-PCR products after restriction enzyme treatment are indicated on the right.
(C) RT-PCR analysis of trans-splicing. Specific amplification of the exon 3-2, exon 1-2, or exon 3-4 junctions, or exon 3 alone was performed with total RNA from the wild-type (WT, lane 1), or the transformed HepG2 cells (lanes 2-7). The products derived from the transgene (T) and from the endogenous Sp1 gene (E) were distinguished using restriction enzymes shown on the right.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

3. Figure 2
Naturally Occurring trans-Splicing between pre-mRNAs from the Genes with a Long Intron or a Pausing Site for RNAPII in an Intron
(A) Partial genomic structures of human genes including the Sp1 gene, the insulin receptor gene and the gene for AK cDNA clone (left). Exons are shown as boxes. The sizes of exons and introns were not drawn on scale. The results of RT-PCR amplification with (+) or without (–) reverse transcription were shown with the sizes of PCR products (right). Structures of the PCR products were identified by DNA sequencing.
(B) The genomic structure of the rat apolipoprotein A-I gene. The positions of the two arrest sites for RNAPII are indicated by arrows (Dallinger et al., 1999). The result of the RT-PCR amplification with (+) or without (–) reverse transcription is shown with the size of the product on the right.
(C) Schematic representation of integrated transgenes (Apo and Apo-Δarrest) that were generated with a genomic DNA fragment of rat apolipoprotein A-I gene and a polyadenylation signal of bovine growth hormone gene. In the Apo-Δarrest construct, the pause site of RNAPII was replaced with an unrelated sequence (a dotted line). The products of RT-PCR amplification for the exon and the exon arrangements were shown with their sizes on the right.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

4. Figure 3
Pausing Sites for RNAPII in an Intron Cause Homotypic trans-Splicing
(A) Schematic representation of the C1-A and C1-MAZBS transgenes. Four copies of the MAZ binding site (MAZBSx4) or an unrelated sequence of similar length were inserted into the third intron of the C1 transgene. The unrelated sequence was obtained from the third intron of the Sp1 gene. Region 1 and 2 represent the two regions used in CHIP.
(B) RT-PCR analysis with total RNA from the cell line expressing the C1-A transgene or the C1-MAZBS transgene. The MAZΔN-FLAG vector (+) or a control vector (–) was transfected. The products derived from the transgene (T) and from the endogenous Sp1 gene (E) were distinguished by restriction enzyme digestion, with enzymes shown on the right.
(C) RT-PCR analysis of the total level of both endogenous and transiently expressed MAZ mRNAs. Similar results were also obtained from different cycles of PCR amplification.
(D) Western blot analysis with anti-FLAG antibody. Only the transiently expressed MAZΔN-FLAG protein can be detected.
(E) CHIP assays with the anti-RNAPII antibody. Regions 1 and 2 were amplified by PCR with specific primers. The relative DNA amounts of regions 1 and 2 in each cell lysate used for CHIP assays were verified by PCR amplification (input). Different numbers of PCR cycles were used for amplification of immunoprecipitated DNA and input DNA samples (see Experimental Procedures).
(F) Relative density of RNAPII at the region 2. The value was estimated from the ratio of intensity of the band for region 2 to that for region 1. Mean ± SEM is given with the results of four independent CHIP assays including the result in (E). The ratio obtained from the C1-A cell line transfected with an empty vector was set to one.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

5. Figure 4 A Possible Model for Homotypic trans-Splicing
In this model, two RNAPIIs are simultaneously transcribing a gene. Each 5′ splice site associates with the CTD from the RNAPII that transcribed it. The probability that the 5′ splice site will dissociate from the CTD while the intron is being synthesized is proportional to the time required for intron synthesis (if there are no RNAPII pausing sites in the intron, then it may be proportional to intron length). Once the 5′ splice site dissociates from the CTD, it can reassociate either with the same CTD or with a CTD of another polymerase already carrying 3′ splice site. In principle, more than one 5′ splice site could associate with a single CTD, as it contains multiple heptad repeats (52 repeats in mammalian RNAPII). The two associated 5′ splice sites could then compete for the single 3′ splice site. trans-splicing would occur if the 5′ splice site from exon 3 joins to the 3′ splice site of exon 2, resulting in the formation of an mRNA with the exon arrangement.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions