Figure 1.
Analysis of the human 5′-UTR database, overview of the approach, and construction of the in-frame cDNA expression library.
(A) Analysis of the human 5′-UTR database (http://utrdb.ba.itb.cnr.it/) to predict their effects on expressed sequences following translation with a YFP1 tag peptide as fusion proteins during the construction of a prey cDNA library. (B) Overview of the screening procedure. (C) For the construction of the in-frame cDNA expression library, mRNA was isolated from normal human urothelial cells and was used as a template for first-strand cDNA synthesis using polyT primer. Double-stranded cDNAs without 5′-UTRs were synthesized using primers 1 and 2 (representing approximately 40% of the Kozak sequences that are present in vertebrate genomes) complemented with primer mixes 3 and 4 (representing the remaining 60% of the Kozak sequence combinations in vertebrates). In primer mixes 3 and 4, the combination of sequences referred to as “D” is an equal mixture of A, G and T, “H” is an equal mixture of A, C and T, “K” is an equal mixture of G and T, and “W” is an equal mixture of A and T. There are 19,683 and 157,464 possible sequence combinations in primer mixes 3 and 4, respectively. (D) Sequence analysis of the in-frame cDNA library was performed on 198 representative plasmids isolated from random colonies of the library.
Figure 2.
Predicted expression of CRABP2 and PGAM1 proteins by the constructs with and without 5′-UTRs.
(A) Comparison of CRABP2 expression constructs with and without the CRABP2 5′-UTR. (B) Comparison of PGAM1 expression constructs with and without the PGAM1 5′-UTR.
Figure 3.
Identification of the CRABP2 and PGAM1 proteins as ARL11-binding partners using the in-frame cDNA expression library.
(A) Western blot analysis of N-terminal YFP1-tagged fusion proteins expressed by the constructs with and without 5′-UTRs in HEK-293T cells. Expression of a construct containing only YFP1 was used as a control. An anti-GFP N-terminal antibody was used to visualize the expressed tagged proteins. (B) YFP fluorescence in HEK-293T cells after the co-transfection of YFP1-CRABP2 with ARL11-YFP2 and YFP1-PGAM1 with ARL11-YFP2. Nuclei were counterstained with DAPI. (C) Confirmation of the interaction between ARL11 and CRABP2 by western blotting and co-immunoprecipitation. HEK-293T cells were transfected with HA-tagged ARL11 and FLAG-tagged CRABP2 constructs without its 5′-UTR. Protein expression was verified by immunoblotting using anti-ARL11 and anti-CRABP2 antibodies in direct western blots (DWB). Immunoprecipitation with western blotting (IPWB) was performed by anti-HA antibody pull-down of ARL11 to detect CRABP2 binding (top panel). Results were confirmed using a complementary approach (HA-tagged CRABP2, anti-HA antibody immunoprecipitation, and anti-ARL11 immunoblotting (bottom panel). (D) Confirmation of ARL 11 and PGAM1 binding by IPWB. HEK-293T cells were transfected with HA-tagged ARL11 and flag-tagged PGAM1 constructs as indicated. Protein expression verified by immunoblotting with anti-PGAM1 (top panel) or anti-ARL11 (bottom panel) antibodies (DWBs). IPWBs were performed by anti-HA immunoprecipiation of ARL11 followed by immunoblotting with anti-PGAM1 (Top panel). Alternatively, immunoprecipitation was performed using HA-tagged PGAM1 followed by immunoblotting with anti-ARL11 (Bottom panel). (E) The in-frame cDNA library prevented interference caused by the CRABP2 5′-UTR that inhibits its binding to ARL11. HEK-293T cells were transfected with HA-ARL11 and with YFP1-CRABP2 or YFP1-5′-UTR-CRABP2 as indicated. Protein expression was confirmed by immunoblotting with anti-CRABP2 antibody (top panel) or anti-YFP1 antibody (bottom panel). Alternatively, ARL11 was immunoprecipitated using the anti-HA antibody, and bound proteins were detected by immunoblotting with anti-CRABP2 (top panel) or anti-YFP1 (bottom panel) antibody. (F) The in-frame cDNA library prevents interference caused by the PGAM1 5′-UTR that prevents its binding to ARL11. HEK-293T cells were transfected with HA-ARL11 and YFP1-PGAM1, or YFP1-5′-UTR-PGAM1. Protein expression was confirmed using anti-PGAM1 (top panel) or anti-YFP1 (bottom panel) antibodies (DWB). To identify ARL11-associated proteins (IPWB), ARL11 was immunoprecipitated using the anti-HA antibody and bound proteins were detected using either an anti-PGM1 (top panel) or anti-YFP (bottom panel) antibody.
Table 1.
Sequences of forward primers used to construct in-frame cDNA library.