Summary: Humans have two nearly identical copies of Survival Motor Neuron (SMN) gene, SMN1 and SMN2. Low SMN levels due to deletion and/or mutation of SMN1 lead to spinal muscular atrophy (SMA), a major genetic disease associated with infant mortality. SMN2 fails to compensate for the loss of SMN1 due to skipping of exon 7. Since the full-length mRNAs of both genes code for identical proteins, correction of SMN2 exon 7 splicing provides one of the best therapeutic options. We discovered Intronic Splicing Silencer N1 (ISS- N1) as a promising therapeutic target for an antisense oligonucleotide (ASO)-mediated correction of SMN2 exon 7 splicing. Nusinersen (SpinrazaTM), an ISS-N1-targeting ASO, was recently approved by the FDA (USA) as the first drug for the treatment of SMA. While nusinersen has been successful in halting deaths of a vast majority of SMA infants and slow the disease progression, problems still persist with respect to the speed and extent of recovery. Hence there is an urgent need to develop alternative and/or complementary mechanism- based therapies for an improved treatment of SMA. This proposal is aimed at understanding the transcription- coupled splicing regulation (TCSR) of the SMN genes to uncover novel therapeutic targets for enhancing SMN levels from SMN2. This project is based on the premise that transcription initiation and elongation regulate both SMN2 transcripts levels and SMN2 exon 7 splicing. Our proposed study will combine several complementary and powerful techniques, including native elongating transcription sequencing (NET-seq), precision nuclear run-on sequencing (PRO-seq), in vivo structure probing and affinity purification of complexes deposited on the nascent RNAs during transcription by RNA polymerase II (pol II). To ensure the feasibility of our study, we have generated a SMN2 “super minigene” comprised of the full-length SMN2 promoter, all exons, their flanking intronic sequences and the 3¢ untranslated region (3¢UTR) of SMN2. We are proposing this study in the light of a related discovery that SMN genes produce a vast repertoire of circular RNAs (cRNAs or circRNAs). In Aim 1, we will test the hypothesis that cis-elements within both the promoter and the transcribed region of SMN2 regulate transcription (initiation and elongation) and consequently influence whether exon 7 will be included or skipped. Employing PRO-seq, we will analyze pol II pause sites during transcription of SMN in different cell types. We will determine if transcription pause sites are involved in TCSR of SMN2 exon 7. We will assess the effect of small molecules, including transcription and splicing modulators, on TCSR of SMN2 exon 7. We will also determine if transcription and splicing modulators affect the generation of SMN cRNAs. We will analyze tissues from SMA mouse models to determine if cRNAs generated by SMN have relevance to SMA pathogenesis. Employing a library of SMN2 super minigenes, we will uncover the role of promoter elemen...