Fig 1.
The first AUG codon in mouse Nelf-b gene is dispensable for full-length protein translation.
(A) Diagram showing various NELF-B expression constructs. “AUG” contains the NELF-B coding sequence starting at the annotated initiating AUG. The solid box denotes the location of the Flag peptide. “FL” contains the entire 5’-UTR sequence of Nelf-b. The “CUC” construct has the mutation in the annotated AUG codon. (B) NELF-B and Flag western blot showing the expression of the constructs described in (A) in 293T cells. Two independent clones of “FL-CUC” and “Flag-FL-CUC” were included.
Fig 2.
Sequence of the first exon of mouse Nelf-b gene.
(A) Codons of interest are highlighted in red (CUG-143), green (other potential translational initiation alternative codons), and blue (annotated initiating AUG codon). (B) RNA folding and hybridization prediction for the 70 bp sequence downstream of CUG-143 in the 5’-UTR of mouse Nelf-b (ΔG = -24.20 Kcal/mol).
Fig 3.
A non-canonical codon is important for translation of full-length NELF-B.
Expression of NELF-B protein with various point mutations at different alternative initiation codons in MEFs.
Fig 4.
The non-AUG initiation codon is conserved among mammals.
(A) Sequence alignment around the CUG-143 and GUG-188 codons. (B) Western blot showing that 5’-UTR of Human NELF-B is required for full-length protein translation.
Fig 5.
Both full-length and AUG-initiated NELF-B are capable of supporting proliferation of mouse embryonic fibroblasts.
(A) Western blots indicating both AUG and FL NELF-B were capable of interacting with the other NELF subunits in MEFs. (B) Growth curve for WT and Nelf-b KO MEFs. (C) Growth curve showing AUG-NELF-B expressing cells in the WT and KO background. (D) Growth curve showing FL-NELF-B expressing cells in the WT and KO background. * p < 0.05.
Fig 6.
Expression of NELF-B in mouse tissues.
Western blots of endogenous NELF-B protein in different mouse adult tissues Aterisks indicate the shorter version of the endogenous protein. 10T1/2: lysate from a mouse adipose progenitor cell line; MDA-MB-231: lysate from a human breast cancer cell line; Flag-AUG and Flag-FL: Flag-tagged mouse NELF-B ectopically expressed in MEF depleted of endogenous NELF-B. Fifty and thirteen μg of total tissue lysates were loaded in the top and bottom gels, respectively.