Abstract
Introduction: Polymorphisms spanning 4q24 are associated with lung function and risk of COPD. The integrator complex subunit 12 gene (INTS12) is a candidate gene in the region for these phenotypes. INTS12 is part of a complex responsible for processing spliceosomal small nuclear RNAs (snRNAs) (1). Human INTS12 shows homology to other proteins involved in gene regulation (2).
Aims: The aim of this study was to optimize INTS12 knockdown in primary human bronchial epithelial cells (HBECs) in order to investigate the function of INTS12 including snRNA processing.
Methods: siRNA delivery to HBECs (passage 3) was optimized by transfecting Cy3-siRNA with INTERFERin followed by epifluorescent imaging. Three siRNA sequences (10nM) targeting human INTS12 were assessed for knockdown of INTS12 expression using qPCR. HPRT1 silencing acted as a positive control. The ratio of immature to total U2 transcripts was assayed by qPCR, to indicate U2 processing.
Results: HPRT1 mRNA expression was attenuated by 90% in HPRT1 siRNA-transfected HBECs (relative to scrambled siRNA transfected cells, n=2). INTS12 mRNA expression in HBECs transfected with INTS12-targeting siRNAs was decreased by 80 ± 6% and 69 ± 3% (p<0.05, n=3) for two siRNAs while not significant for the third siRNA. Preliminary data indicate that maximal INTS12 knockdown has a functional effect on U2 processing and results in ∼60-fold accumulation of immature U2 as assessed by the ratio of primary/total U2 (n=2).
Conclusions: INTS12 is likely to play an important role in HBEC function via snRNA processing: this may affect transcription globally.
Funding: Medical Research Council
1. Baillat et al. (2005) Cell 123 (2): 265–76.
2. Obeidat et al. (2013) PLoSOne 18;8(9): e74630.
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