1
Data S1
S1, Details of clonings
To generate UPF1 N-terminal mapping constructs, PCR fragments were amplified using
DreamTaq Green PCR Master Mix (Thermo Scientific, # K1082) from different UPF1
constructs (see below the details of constructs, primers and templates) and cloned into BamHI
digested ΔNΔC UPF1 vector. The correct orientation was tested by PCR and the selected
clones were sequenced.
To create U1ΔC construct, the complete N-terminal domain was amplified with U1NterF1 for, U1N-terF4 rev primers, and then the fragment was digested and incorporated into
BamHI cleaved ΔNΔC vector.
The mutant N-terminal fragments were generated by PCR mutagenesis. To generate
NP3A, mutagenized N-terminal fragment was generated with U1N-terF1 for, U1-F3 ala
cserelo R; U1-F3 ala cserelo F, U1N-terF4 rev primers, and then the fragment was cleaved
and cloned into ΔNΔC vector. Similar cloning strategy was used to generate NP1-2A4A,
NP1-4A, NΔNn, NΔNc, NΔNcP3A, NΔNcP1-2A, NΔcP1-3A, NΔP1-2, NΔP1-2P3A and
NΔP1-2P3D UPF1 N-terminal mapping constructs.
The C-terminal mapping constructs were PCR amplified, cleaved with AvrII and
XhoI, and then the cleaved fragments were cloned into AvrII and SalI digested U1ΔN vector,
thereby replacing the whole C-terminal domain of the U1ΔN construct. To map the C1 region,
PCR mutagenized C1 fragments were generated, cleaved and cloned into AvrII-SalI digested
U1ΔN vector.
To analyze co-localization and interaction, cDNAs of SMG7, UPF1, DCP1 were amplified
from total RNA sample that was isolated from wild-type Arabidopsis plant using Phusion
High-Fidelity DNA Polymerase (Thermo Scientific). The cDNAs were cloned into
pENTR/D-TOPO (SMG7) and pDONR201 (UPF1, DCP1), obtaining pENTR/DTOPOSMG7, pDONR-UPF1 and pDONR-DCP1 plasmids. LR reaction (Invitrogen) was then
performed between pGD120-GW-sCFP3A and pENTR/DTOPO-SMG7, between pGD120GW-sYFP2 and pDONR-UPF1 and between pGWB554 (Nakagawa et al., 2009) and
pDONR-DCP1 resulting in S7-C U1-Y and D1-R constructs.
To generate U1ΔC-Y and NP1-4A-Y constructs, PCR fragments were amplified from U1ΔC
and NP1-4A constructs with UPF1dTOPOf-UPF1dCokdTOPOr and F1AUPFdTOPOfUPF1dCokdTOPOr primer pairs, respectively, and then the fragments were cloned into
pENTR/D-TOPO vector. The obtained constructs were used in LR reaction with pGD120GW-sYFP2 plasmid.
2
To generate full length NP1-4A-U1-Y construct, U1-Y plasmid were cleaved with Bsu361
and XhoI and then the cleaved fragments were cloned into Bsu361 and XhoI digested NP14A-Y vector, thereby cloning the whole C-terminal domain of the U1-Y construct.