The 5′‐UTR of Niemann‐Pick C1‐Like 1 Enhances Translation Efficiency
Authors: Ticho Alexander L , Malhotra Pooja , Manzella Christopher , Saksena Seema , Gill Ravinder K , Dudeja Pradeep K , Alrefai Waddah A 
Published: November 2023

Intestinal Neimann‐Pick C1 Like‐1 (NPC1L1) is essential for cholesterol absorption. Reduction in NPC1L1 expression reduces plasma cholesterol and lowers the risk of atherosclerosis. Gene expression may be modulated by transcriptional and/or post‐transcriptional mechanisms. Previous studies have demonstrated that NPC1L1 transcription is regulated by transcription factors, as well as by CpG methylation of the NPC1L1 promoter. However, the mechanisms underlying the basal transcription and translation of NPC1L1 are still not well understood. This study aims to understand the mechanisms responsible for basal expression of NPC1L1 and to investigate the potential role of the 5′ Untranslated Region (5′UTR) in modulating the translation efficiency of NPC1L1. A region of DNA upstream of the NPC1L1 coding sequence (‐1741/+56, +1 is the transcription initiation site) was cloned into a promoterless pCpGfree‐basic‐Lucia vector and designated as L1. This construct was transfected into intestinal Caco2 cells by electroporation, and Lucia activity was measured 48 h post‐transfection. The L1 construct displayed robust promoter activity above the empty vector (131 ± 33 fold, p < 0.001). Progressive 5′ deletions L2 (−1091/+56), L3 (−291/+56), L4 (−244/+56), and L5 (−135/+56) showed no significant change in the reporter activity (L5: 0.85 ± 0.11 fold relative to L1, ns), indicating that L5 fragment contains the basal promoter of NPC1L1 gene. In vitro CpG methylation of all constructs showed a significant reduction in reporter activity suggesting that the suppressive effect of DNA methylation is not sequence‐specific. A construct with 3′ deletion ‐1741/‐51 displayed low reporter activity relative to L1 (6.7 ± 2.8 %, p < 0.0001). Further deletions up to ‐1741/‐238 displayed similar low levels of reporter activity. Along with the results from the 5′ deletions, these findings indicate that the −51/+56 region harbors the minimal promoter of the NPC1L1 gene. Interestingly, deletion of the 5′UTR region (−1741/+1) decreased the reporter activity by 53 ± 10% (p < 0.05) without altering the mRNA levels of the reporter gene (lucia). These data suggest that deletion of the 5′ UTR decreased reporter activity by suppressing translation efficiency. We next generated a plasmid containing an SV40 promoter and the NPC1L1 5′ UTR upstream of luciferase. Reporter activity was increased by 3.08 ± 0.60 fold (p < 0.05) relative to the empty vector lacking a 5′ UTR, with no difference in mRNA levels of the reporter gene (1.14 ± 0.19 fold relative to empty vector, ns), confirming the role of NPC1L1 5′ UTR in enhancing translation efficiency. In conclusion, our data have localized the minimal NPC1L1 promoter to the region between −51 and ‐1 in the NPC1L1 gene and demonstrated that the NPC1L1 5′ UTR is crucial for proper mRNA translation efficiency. Delineating the molecular basis for basal NPC1L1 transcription and translation may unravel potential therapeutic targets to decrease NPC1L1 expression and lower the levels of plasma cholesterol.

Support or Funding Information

NIH R01: DK71596 and DK109709, VA merit review BX000152