1. Volume 10, Issue 1, Pages 21-33 (July 2002)
Promoter Choice Determines Splice Site Selection in Protocadherin α and γ Pre-mRNA Splicing 
Bosiljka Tasic, Christoph E. Nabholz, Kristin K. Baldwin, Youngwook Kim, Erroll H. Rueckert, Scott A. Ribich, Paula Cramer, Qiang Wu, Richard Axel, Tom Maniatis 
Molecular Cell 
Volume 10, Issue 1, Pages (July 2002)
DOI: /S (02)

2. Figure 1
The Organization of Mouse Pcdh Gene Clusters and Models for Pcdh Gene Expression
(A) The linked α, β, and γ gene clusters span approximately 900 kb of mouse chromosome 18. Splicing of V exons to constant exons generates 58 and 53 Pcdh isoforms in mouse and human, respectively. Alternative splicing within the last α constant exon increases the number of protein isoforms to 72 and 68 (Sugino et al., 2000). The V exons are indicated as ovals and constant region (C) exons as rectangles. The α, β, and γ cluster exons are black, white, and gray, respectively; the c-type V exons (c1 and c2 in α, and c3, c4, and c5 in γ cluster) are striped.
(B) Three models for cell-specific expression of Pcdh isoforms. Each model is illustrated by the corresponding pre-mRNA(s). Gray lines represent cis-splicing, dashed gray lines trans-splicing, and black half-circles the 7-methyl guanosine caps. Model 1, single promoter is located upstream of a noncoding 5′ leader exon, which alternatively cis-splices to selected V exons. Only the V exon spliced to the leader exon further splices to the first constant exon. Model 2, each V exon is preceded by a promoter. Only the active promoter-proximal V exon cis-splices to the first constant exon. Model 3, each V exon and the first constant exon are preceded by a promoter. V and constant exons from separate pre-mRNAs trans-splice.
Molecular Cell  , 21-33DOI: ( /S (02) )

3. Figure 2
Each Pcdh V Exon Is Regulated by a Promoter Containing a Conserved Sequence Element
(A) Identification of transcription start sites for Pcdh mRNAs by 5′ RACE. The identified transcription start sites are shown as small vertical lines. The numbers above represent the number of individual clones obtained for the particular start site. The analyzed mouse (M) and human (H) V exons are marked using the color and shape exon code as in Figure 1. CSEs are shown as gray boxes.
Molecular Cell  , 21-33DOI: ( /S (02) )

4. Figure 3
RT-PCR/Allele-Specific Restriction Digestion Assay for Interchromosomal trans-Splicing in M/S F1 Brain RNA
DNA fragments obtained after restriction digestion by HinfI and BamHI of γb7/γ constant RT-PCR products from different mouse strains were analyzed by polyacrylamide gel electrophoresis. The labeling of each lane corresponds to the mouse strain (M, S, or M/S F1) from which total RNA was isolated; M + S, equal amounts of M and S cDNAs mixed before the PCR; U is the same as M, but undigested; MW, molecular weight marker. All of the possible γb7/γ constant RT-PCR products are illustrated on the right with the alleles designated as (M) and (S). The positions of allele-specific restriction sites are shown as vertical white bars. The fragments that contain the splice junction, and their lengths obtained after restriction digestion, are designated below each RT-PCR product. As M/S band overlaps with the undigested band present in all the lanes, this precludes the estimation of the abundance of hybrid M/S mRNA.
Molecular Cell  , 21-33DOI: ( /S (02) )

5. Figure 4
Inversion of α7 Exon in the Pcdh α Cluster Abolishes Its Splicing to α Constant Exons
(A) A schematic diagram of the strategy used to invert the orientation of the α7 V exon in the α cluster. Top to bottom: the genomic organization of a part of the α cluster variable region; targeting vector with the inverted α7 exon (α7F); α cluster after homologous recombination; α cluster after Cre-mediated site-specific recombination (α7FC). The orientation of transcription is indicated by white arrows. “LTNL,” selection cassette containing thymidine kinase and neo resistance genes flanked by two loxP sites (black triangles). Restriction sites B, BstXI; H, HindIII; M, MfeI; X, XhoI. The gray box designated “probe” is the Southern blot probe used in (B).
(B) Confirmation of correct α7 inversion by HindIII restriction digestion of ES cell genomic DNA, followed by Southern blotting. The 4.6 kb and 6.7 kb bands correspond to the α7F and α7FC alleles, respectively. The 5.2 kb band represents the wild-type allele.
(C) Analysis of RNAs from wt (+/+), α7F/+, and α7FC/+ ES cells for the presence of the pre-mRNAs containing α7 V exons. RT-PCR products were obtained using α7V exon-specific primers and were subsequently digested with restriction enzymes XhoI (X; α7F- and α7FC-specific) or BstXI (B; α7wt-specific). Expected restriction fragments for all alleles and their lengths in base pairs are illustrated below the gel. U, undigested PCR product; MW, molecular weight marker.
(D) Analysis of RNAs from wt (+/+), α7F/+, and α7FC/+ ES cells for the presence of the mRNAs containing α7 V exons spliced to α constant exons. RT-PCR products were obtained using an α7V exon-specific primer and an α constant primer and were subsequently digested with same restriction enzymes as in (C). Expected restriction fragments for all alleles with their lengths in base pairs are illustrated below the gel. U, undigested PCR product; MW, molecular weight marker; SJ, splice junction.
Molecular Cell  , 21-33DOI: ( /S (02) )

6. Figure 5 Detection of Intercluster Hybrid Pcdh mRNAs
(A) A schematic diagram of the three Pcdh clusters with an example of a trans-spliced intercluster mRNA containing a Pcdh γV exon and α constant exons shown below.
(B) Detection of intercluster Pcdh RNAs by RT-PCR of mouse brain total RNA. The analyzed hybrid RNAs are schematically illustrated above each gel. Top and bottom gels show γV/α constant and αV/γ constant RT-PCR products, respectively. The order of loading corresponds to the 5′ to 3′ order of V exons on mouse chromosome 18. Under these PCR conditions, correct PCR products were not detected for γa7, γb4, γa8, γb6, γb7, γb8, γc4, γc5 splicing to α constant, and αc2 splicing to γ constant. MW, molecular weight marker.
(C) Comparison of Pcdh intracluster and intercluster RNA splicing patterns in the mouse Neuro-2a cell line by RT-PCR. Top and bottom gels show intracluster and intercluster RT-PCR products, respectively. The mRNAs detected are schematically illustrated above each gel. All primer combinations gave correct RT-PCR products on mouse brain total RNA (data not shown). The lanes are marked with V exon identities, as in (B). MW, molecular weight marker.
(D) Analysis of intercluster and intracluster Pcdh mRNAs by RNase protection assay. The probes were hybridized to mouse brain total RNA (200 μg and 50 μg for intercluster and intracluster RNAs, respectively) or the corresponding amounts of yeast tRNA. Lane T, riboprobe incubated with mouse brain total RNA after RNase A/T1 digestion; Lane C, the same as lane T but riboprobe incubated with yeast tRNA; Lane U, an aliquot of riboprobe incubated with mouse brain total RNA before RNase A/T1 digestion. The probes and the protected fragments with the corresponding lengths are illustrated at the sides of the gels (P, vector sequence).
Molecular Cell  , 21-33DOI: ( /S (02) )

7. Figure 6 Detection of Intergenic mDia1/Pcdh trans-Spliced RNAs
(A) A schematic diagram of intergenic trans-splicing between mDia1 and Pcdh constant exons. The pre-mRNAs containing γ constant or mDia1 are transcribed from opposite DNA strands as illustrated by the wavy arrows. The detected intergenic RT-PCR products are illustrated below. trans-splicing occurs between exon 18 or 27 of mDia1 and different exons from α and γ constant regions. The hybrid cDNAs that maintain the reading frame of constant exons are indicated by asterisks.
(B) Analysis of mDia1 exon 27/Pcdh γ constant exon 3 hybrid RNA by RNase protection assay. Lane T, riboprobe incubated with 100 μg of mouse brain total RNA after RNase A/T1 digestion; Lane C, the same as lane T but the riboprobe was incubated with 100 μg yeast tRNA; Lane U, an aliquot of the riboprobe incubated with mouse brain total RNA before RNase A/T1 digestion; MW, molecular weight marker. The probe and the protected fragments with the corresponding lengths are illustrated on the right side of the gel (P, vector sequence).
Molecular Cell  , 21-33DOI: ( /S (02) )