Analysis of plasmid-based SMN transcripts. (A) RT-PCR amplification of total RNA isolated from neuroblastoma C6 cells 48 h posttransfection with plasmid-based SMN1 (pSMN1), SMN2 (pSMN2), vector alone (pVector), or mock-transfected vector by using Lipofectamine (Life Technologies). FL SMN and SMNΔ7 transcripts are indicated and have been sequenced to ensure fidelity of splicing events. Two vector transcript species are a result of incomplete excision of the plasmid-based intron. No reverse transcriptase (-RT pSMN1) served as a control. (B) pSMN1 or pSMN2 (100 ng, 500 ng, 1 μg, 5 μg, and 10 μg) was transiently transfected into C6 cells. RT-PCR was performed (see A), and FL SMN and SMNΔ7 transcripts are indicated. (C) Transient expression of pSMN1 or pSMN2 in C33A (cervical carcinoma), C6 (neuroblastoma), T98G (glioblastoma), and Cath.a and CAD (murine neuroblastoma) cell lines and RT-PCR analysis of plasmid SMN transcripts (see A). (D) Synthetic SMN1 hybrid constructs. Two SMN2 nucleotides were introduced into a pSMN1 construct (In, intron; Ex, exon): pSMN1ΔIn6/Ex7; -ΔEx7/In7+100; -ΔEx7/In7+214. SMN2-derived nucleotides follow “Δ.” (E) Single SMN2 nucleotides introduced into pSMN1: pSMN1ΔEx7; -ΔIn6; -ΔIn7+100; -ΔIn7+214. SMN2-derived nucleotides follow “Δ.” (F) Single SMN1 nucleotides introduced into pSMN2: pSMN2ΔIn6; -ΔEx7; -ΔIn7+100. SMN2-derived nucleotides follow “Δ.” A 100-bp ladder is indicated.

Endogenous SMN hybrid gene transcript analysis. (A) Expanded view of the 3′ region of SMN genes. Intron (lines) and exon (boxes) boundaries are indicated. Positions of the five nucleotide differences between SMN1 and SMN2 within intron 6 (In6, G/A), exon 7 (Ex7, C/T), intron 7 (In7+100, A/G; In7+214, A/G), and exon 8 (Ex8, G/A) are shown (SMN1/SMN2 sequence). The new intron 7 mutation (*In7 +6), and splice elements are shown (3′ ss, 3′ splice site; 5′ ss, 5′ splice site; PolyPy Tract, polypyrimidine tract). (B) RT-PCR amplification of SMN transcripts from Epstein–Barr virus transformed from SMA and control individuals by using oligonucleotides located in SMN exons 5 and 8. Products were digested with DdeI and resolved in a 2% agarose gel. The SMN2 nucleotide within exon 8 creates a DdeI site, resulting in faster-migrating species for SMN2 transcripts. The positions of full-length (FL) and exon 7-skipped (Δ7) transcripts are indicated. Graphic representations of the 3′ end of the hybrid genes and the origin of the five nucleotide polymorphisms are indicated (1, SMN1; 2, SMN2; In, intron; Ex, exon): Hybrid #1 (In6/SMN2, Ex7/SMN2, In7+100/SMN1, In7+214/SMN1, Ex8/SMN1); Hybrid #2a/b (In6/SMN2, Ex7/SMN2, In7+100/SMN2, In7+214/SMN2, Ex8/SMN1). (C) RT-PCR amplification of SMN transcripts from primary fibroblasts and EBV-transformed lymphocytes from SMA and control individuals (see B).

SMNΔ7 production from a novel SMN1 spice-site mutation. RT-PCR analysis (see Fig. 1B) of endogenous SMN transcripts from SMA and control individuals’ EBV-transformed lymphocytes. The father is carrier-ΔSMN1, and the mother is carrierΔc.922+6 T/G. Each has an additional, single SMN1 copy. The patient carries an SMN1 deletion and an SMN1 copy with the splice-site mutation. The SMN1-derived, exon-skipped transcript is shown in lanes 5 and 6 (*Δ7).