Template switching-like RT-PCR products associated with inverted Alu repeats

Alu elements are the most abundant class of short interspersed nuclear elements in the human genome and can influence alternative splicing, particularly when paired Alu elements are located in inverted orientations within introns. However, how inverted Alu-derived RNA structures are reflected in reverse transcription polymerase chain reaction (RT-PCR) products generated during minigene-based splicing analyses remains unclear. Here, we used an ACAT1 minigene model containing inverted AluSx elements to examine RT-PCR products derived from pre-mRNA-containing transcripts. Complementary DNA (cDNA) was synthesized using a minigene-derived primer and amplified with intron-targeting primers spanning the intronic AluSx-containing region. The Inverted AluSx construct generated heterogeneous RT-PCR products, and Sanger sequencing of cloned RT-PCR products identified seven distinct deletion patterns. Each deletion junction was associated with a short microhomology of 2-6 nucleotides. RNAfold analysis predicted that the Inverted AluSx transcript has a greater propensity to form an extended intramolecular duplex-like structure than control transcripts. Together, these findings support a model in which predicted inverted AluSx-derived RNA structures may predispose transcripts to template switching-like events during cDNA synthesis, generating truncated cDNA products that should be distinguished from genuine RNA isoforms in cDNA-based transcript analyses.