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PAHAL is expressed as a sense lncRNA transcript within the PAH locus.

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posted on 29.04.2020, 17:47 authored by Xia Zhang, Ya'nan Xu, Bing Chen, Le Kang

(A) Schematic representation of the catecholamine metabolic pathway. PAH catalyses the reaction from phenylalanine to tyrosine. PAH, phenylalanine hydroxylase; TH, tyrosine hydroxylase; TYDC, tyrosine decarboxylase; TβH, tyramine β-hydroxylase; DDC, decarboxylase. (B) Gene and transcript structure of the locust PAH and PAHAL. The bent arrows indicate the transcriptional orientation of PAH and PAHAL, respectively. “E 1–8” represents the eight exons of PAH transcript. The half-arrows indicate the following strand-specific primers for quantitative PCR (qPCR): primers 1 and 2 for PAH, primers 3 and 4 for PAHAL, primers 5 and 6 for PAHAL dsRNA, and primers 7 and 8 for PAH dsRNA. The broken line represents siRNA sequences used for PAHAL knockdown. The entire PAH locus is drawn proportional to its length. The black scale bar for PAH locus represent 1 kb. The gene structure of PAHAL is scaled up in red. The red scale bar for PAHAL represent 0.1 kb. (C) PAHAL and PAH were different transcripts. The half-arrows indicate the following strand-specific primers for PCR: Primer 13 is the specific reverse primer for cDNA synthesis of PAH and PAHAL, primers 10 and 12 are for PAH (289 bp), primers 9 and 10 are for PAHAL (476 bp), and primers 11 and 12 yield no PCR product. The red shading indicates the location of PAHAL in PAH locus. (D) 5′-triphosphate cap of PAHAL validated through the 5′ exonuclease digestion assay. GAPDH and 18S rRNA acted as the positive and negative controls, respectively. “Total” means total RNA in reverse transcription (RT), and “capped” indicates capped RNA that was 5′ exonuclease digested in the cDNA synthesis. (E) Detection of PAHAL poly(A) tail by RT-PCR in the brain RNA of the fourth-stadium nymphs. Total RNA was G/I-tailed, reverse-transcribed and amplified with gene-specific primers F and R. U6 and GAPDH were used as negative and positive control, respectively. (F) Coding potential of the transcript in the PAHAL and reference transcripts according to the CPAT algorithm. In the references, MALAT1 and HOTAIR are lncRNAs, and the others are protein-coding genes. (G) In vitro translation assay for PAHAL. Rluc, biotinylated luciferase; NC, no DNA-template control.