Pbit1.1 n

The following plasmids were used as received from Addgene: mRuby-Golgi-7 (GalT; #55865), mRuby2-Rab5a-7 (#55911), mCherry-Rab7a-7 (#55127), mCherry-Rab11a-7 (#55124), pmCherry-2xFYVE (#140050). Venus-2xFYVE was made by replacing mCherry in pmCherry-2xFYVE with Venus using NheI and BsrGI. mRuby2-MOA was made by replacing Venus in Venus-MOA using NheI and BglII. mRuby2-PTP1b was made by replacing Venus in Venus-PTP1b using NheI and BsrGI. CMV-LgBit was made by amplifying LgBit from pBiT1.1-N (Promega) and ligating into pcDNA3.1 (+) using HindIII and XhoI. SNAPf-β₂AR, SNAPf-D2R, SNAPf-M3R and D2S-Nluc were kindly provided by Jonathan Javitch (Columbia University). Venus-Kras, Venus-PTP1b, Venus-MOA, Venus-rab5a, Venus-rab7a, Venus-rab11a and memGRKct-Venus were described previously (Hollins et al., 2009 ; Lan et al., 2012 (link)). All plasmids were verified by automated sequencing.

Fusion constructs of mouse Rbm15 and Wtap to NanoBiT subunits were generated as follows: Full-length Rbm15- and Wtap-coding sequences were amplified with the oligonucleotides indicated in Supplemental Table 2 from poly-A-selected mRNA using NEBNext High-Fidelity 2X PCR master mix (New England BioLabs). Overhangs with homology to destination vectors (pBiT1.1-C, pBiT2.1-C, pBiT1.1-N, and pBiTN.1-C; Promega) were included in oligonucleotide sequences. Gel-purified PCR products were cloned into EcoRI sites using NEBuilder HiFi DNA assembly kit (New England BioLabs) following the manufacturer's recommendations. The optimal combinations of N-terminal- or C-terminal-tagged fusions to small or large subunits were determined through transfection of 20,000 wild-type mESCs per well with Lipofectamine 3000 reagent (Invitrogen) seeded in 96-well tissue culture plates (Corning, catalog no. 3917). Measurements were performed using the Nano-Glo live-cell assay system (Promega) and measured in a microplate luminometer (Berthold, LB960). The Rbm15/Wtap fusion combination yielding the highest luciferase activity was then transfected into distinct mESC genetic backgrounds, and the expression level of the fusion construct was quantified via RT-qPCR using oligonucleotides described in Supplemental Table 2.

All polymerase chain reaction (PCR) amplification and site-directed mutagenesis, including point and deletion mutations were performed using Platinum™ Pfx or SuperFi™ DNA polymerase (Thermo Fisher Scientific). Subcloning of open reading frames (ORFs) and their derivatives into expression plasmids including pICE (a gift from Steve Jackson; Addgene plasmid #46960), pGEX-4T-1 (GE Healthcare Life Sciences), and NanoBiT® system vectors (Promega) was performed using appropriate restriction enzyme sites (Supplementary Table 1). Overlap extension PCR^{56 (link)} was carried out for replacement of the NIX tail-anchor (TA) region with the TA region of other tail-anchored proteins and the fused genes were cloned into vectors pBiT1.1-N (Promega) and pICE_V5^{33 (link)}. Primers are listed in Supplementary Table 3.