In-Frame cDNA Library Combined with Protein Complementation Assay Identifies ARL11-Binding Partners

<div><p>The cDNA expression libraries that produce correct proteins are essential in facilitating the identification of protein-protein interactions. The 5′-untranslated regions (UTRs) that are present in the majority of mammalian and non-mammalian genes are predicted to alter the expression of correct proteins from cDNA libraries. We developed a novel cDNA expression library from which 5′-UTRs were removed using a mixture of polymerase chain reaction primers that complement the Kozak sequences we refer to as an “in-frame cDNA library.” We used this library with the protein complementation assay to identify two novel binding partners for ras-related ADP-ribosylation factor-like 11 (ARL11), cellular retinoic acid binding protein 2 (CRABP2), and phosphoglycerate mutase 1 (PGAM1). Thus, the in-frame cDNA library without 5′-UTRs we describe here increases the chance of correctly identifying protein interactions and will have wide applications in both mammalian and non-mammalian detection systems.</p> </div

Identification of the CRABP2 and PGAM1 proteins as ARL11-binding partners using the in-frame cDNA expression library.

<p>(<b>A</b>) 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>B</b>) YFP fluorescence in HEK-293T cells after the co-transfection of YFP1-<i>CRABP2</i> with <i>ARL11</i>-YFP2 and YFP1-<i>PGAM1</i> with <i>ARL11</i>-YFP2. Nuclei were counterstained with DAPI. (<b>C</b>) Confirmation of the interaction between ARL11 and CRABP2 by western blotting and co-immunoprecipitation. HEK-293T cells were transfected with HA-tagged <i>ARL11</i> and FLAG-tagged <i>CRABP2</i> 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). (<b>D</b>) Confirmation of ARL 11 and PGAM1 binding by IPWB. HEK-293T cells were transfected with HA-tagged <i>ARL11</i> and flag-tagged <i>PGAM1</i> 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). (<b>E</b>) The in-frame cDNA library prevented interference caused by the <i>CRABP2</i> 5′-UTR that inhibits its binding to ARL11. HEK-293T cells were transfected with HA-<i>ARL11</i> and with YFP1-<i>CRABP2</i> or YFP1-5′-UTR-<i>CRABP2</i> 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. (<b>F</b>) The in-frame cDNA library prevents interference caused by the <i>PGAM1</i> 5′-UTR that prevents its binding to ARL11. HEK-293T cells were transfected with HA-<i>ARL11</i> and YFP1-<i>PGAM1</i>, or YFP1-5′-UTR-<i>PGAM1</i>. 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.</p

Predicted expression of CRABP2 and PGAM1 proteins by the constructs with and without 5′-UTRs.

<p>(<b>A</b>) Comparison of <i>CRABP2</i> expression constructs with and without the <i>CRABP2</i> 5′-UTR. (<b>B</b>) Comparison of <i>PGAM1</i> expression constructs with and without the <i>PGAM1</i> 5′-UTR.</p

Analysis of the human 5′-UTR database, overview of the approach, and construction of the in-frame cDNA expression library.

<p>(<b>A</b>) Analysis of the human 5′-UTR database (<a href="http://utrdb.ba.itb.cnr.it/" target="_blank">http://utrdb.ba.itb.cnr.it/</a>) 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>B</b>) Overview of the screening procedure. (<b>C</b>) 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. (<b>D</b>) Sequence analysis of the in-frame cDNA library was performed on 198 representative plasmids isolated from random colonies of the library.</p