Figure 3.
Figure 3. Clonal mutations of PRDM1 in DLBCL clinical cases. (A) Expression analysis of PRDM1 transcripts in DLBCL by RT-PCR. (i) PCR amplification was performed on cDNAs using specific primers located in exons 2 and 4 (Table 1), which normally generate a 355-bp band upon agarose-gel electrophoresis. PCR amplicons of altered sizes were seen in most cases (1, 2, 5, 6, 7, 8), indicative of splicing aberrations. Note the absence of normal-sized bands in these lymphomas, which are consistent with the lack of expression of the normal PRDM1 transcripts. The relatively weak normal-sized bands seen in tumor no. 7 may be due to the presence of contaminating normal cells that express PRDM1. (ii) Using primers spanning exons 5 and 7, PCR amplification generated 2 major bands of approximately equal intensity in case no. 4, one normal-sized (471 bp), and the other larger than normal (520 bp). Upon sequencing, the normal-sized band is found also to contain a small amount of a slightly larger product (488 bp) in addition to the normal-sized product. The presence of abnormally sized products indicates splicing aberrations. (B) Splicing aberrations associated with point mutations at the exon 2/intron 2 junction. A schematic diagram illustrating splicing abnormalities in tumors 5, 6, 7, and 8. Point mutations at the consensus splice donor sequence (underlined) at the exon 2/intron 2 junction (G → A at position –1 in tumors 5, 7, and 8; t → g at position +2 in tumor no. 6) are indicated in bold. Right: Diagrammatic depiction of splicing abnormalities in tumor no. 1 as a result of RNA editing. Point mutation at the consensus splice donor sequence (underlined) at the exon 2/intron 2 junction (G → A at position –1) is indicated, along with the sequence chromatogram of cDNA showing the editing G-to-A conversion absent in genomic DNA. The cryptic splice donor site actually used in the tumors is in italics. The 103-bp intron 2 sequence inserted in the tumor transcripts and the resulting premature translation termination are marked. The predicted structures of PRDM1α and PRDM1β derived from the respective transcripts are shown, along with their functional domains. (C) A schematic diagram illustrating RNA splicing aberrations associated with point mutation at the exon 3/intron 3 junction, as seen in tumor no. 2. Point mutation (g → t) in position +1 of the consensus splice donor sequence (underlined) is shown in bold. Exon 3 skipping and retention of intron 3 sequence (of unknown length) are indicated. The former splicing abnormality causes an internal deletion in the PR domain, and the latter results in premature translation termination. The predicted structures of PRDM1α and PRDM1β are shown, along with their functional domains. (D) (i) cDNA nucleotide sequence of tumor transcripts retaining intron 2 sequence. The point mutations that inactivate the consensus splice donor site (underlined) are indicated in bold (G → A for tumors 1, 5, 7, and 8; t → g for tumor no. 6). The intron 2 cryptic splice donor site and the splice acceptor site at the intron 2/exon 3 junction used in the tumors are in italics. The retained 103-bp intron 2 sequence and the distance between its 3′ end and the 5′ end of exon 3 (7062 bp) is indicated. The premature translation termination codon UGA is in bold, and the novel peptide sequence predicted from translation of the intron 2 sequence is shown in italics. (ii) Partial cDNA nucleotide sequence showing exon 3 skipping or intron 3 sequence retention in transcripts from tumor no. 2. The point mutation (g → t) at the first nucleotide of intron 3 that inactivates the consensus splice donor site (underlined) is shown. The splice donor and acceptor sites used in splicing between exon 2 and exon 4 (or exon 3) are in italics, and the exon 3 nucleotide sequence and encoded amino acids that are internally deleted due to exon 3 skipping are boxed. Sizes of intron 2 and 3 are shown. The intron 3 sequence of indeterminate length retained in some of the tumor transcripts is partially shown, including the premature termination codon (in bold). The novel peptide sequence translated from the intron 3 sequence is shown in italics.

Clonal mutations of PRDM1 in DLBCL clinical cases. (A) Expression analysis of PRDM1 transcripts in DLBCL by RT-PCR. (i) PCR amplification was performed on cDNAs using specific primers located in exons 2 and 4 (Table 1), which normally generate a 355-bp band upon agarose-gel electrophoresis. PCR amplicons of altered sizes were seen in most cases (1, 2, 5, 6, 7, 8), indicative of splicing aberrations. Note the absence of normal-sized bands in these lymphomas, which are consistent with the lack of expression of the normal PRDM1 transcripts. The relatively weak normal-sized bands seen in tumor no. 7 may be due to the presence of contaminating normal cells that express PRDM1. (ii) Using primers spanning exons 5 and 7, PCR amplification generated 2 major bands of approximately equal intensity in case no. 4, one normal-sized (471 bp), and the other larger than normal (520 bp). Upon sequencing, the normal-sized band is found also to contain a small amount of a slightly larger product (488 bp) in addition to the normal-sized product. The presence of abnormally sized products indicates splicing aberrations. (B) Splicing aberrations associated with point mutations at the exon 2/intron 2 junction. A schematic diagram illustrating splicing abnormalities in tumors 5, 6, 7, and 8. Point mutations at the consensus splice donor sequence (underlined) at the exon 2/intron 2 junction (G → A at position –1 in tumors 5, 7, and 8; t → g at position +2 in tumor no. 6) are indicated in bold. Right: Diagrammatic depiction of splicing abnormalities in tumor no. 1 as a result of RNA editing. Point mutation at the consensus splice donor sequence (underlined) at the exon 2/intron 2 junction (G → A at position –1) is indicated, along with the sequence chromatogram of cDNA showing the editing G-to-A conversion absent in genomic DNA. The cryptic splice donor site actually used in the tumors is in italics. The 103-bp intron 2 sequence inserted in the tumor transcripts and the resulting premature translation termination are marked. The predicted structures of PRDM1α and PRDM1β derived from the respective transcripts are shown, along with their functional domains. (C) A schematic diagram illustrating RNA splicing aberrations associated with point mutation at the exon 3/intron 3 junction, as seen in tumor no. 2. Point mutation (g → t) in position +1 of the consensus splice donor sequence (underlined) is shown in bold. Exon 3 skipping and retention of intron 3 sequence (of unknown length) are indicated. The former splicing abnormality causes an internal deletion in the PR domain, and the latter results in premature translation termination. The predicted structures of PRDM1α and PRDM1β are shown, along with their functional domains. (D) (i) cDNA nucleotide sequence of tumor transcripts retaining intron 2 sequence. The point mutations that inactivate the consensus splice donor site (underlined) are indicated in bold (G → A for tumors 1, 5, 7, and 8; t → g for tumor no. 6). The intron 2 cryptic splice donor site and the splice acceptor site at the intron 2/exon 3 junction used in the tumors are in italics. The retained 103-bp intron 2 sequence and the distance between its 3′ end and the 5′ end of exon 3 (7062 bp) is indicated. The premature translation termination codon UGA is in bold, and the novel peptide sequence predicted from translation of the intron 2 sequence is shown in italics. (ii) Partial cDNA nucleotide sequence showing exon 3 skipping or intron 3 sequence retention in transcripts from tumor no. 2. The point mutation (g → t) at the first nucleotide of intron 3 that inactivates the consensus splice donor site (underlined) is shown. The splice donor and acceptor sites used in splicing between exon 2 and exon 4 (or exon 3) are in italics, and the exon 3 nucleotide sequence and encoded amino acids that are internally deleted due to exon 3 skipping are boxed. Sizes of intron 2 and 3 are shown. The intron 3 sequence of indeterminate length retained in some of the tumor transcripts is partially shown, including the premature termination codon (in bold). The novel peptide sequence translated from the intron 3 sequence is shown in italics.

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