3.6 CRIR1 interacts with MeCSP5 protein
In order to further investigate the biological function of CRIR1 ,
we performed in vitro RNA pull-down assays and filtered the
protein interactome of CRIR1 using mass spectrometry (Figure 6a).
A total of 15 proteins, represented by two or more unique peptides,
specifically interacted with biotin-labeled sense-CRIR1 but not
the control antisense-CRIR1 (Figure 6b). Among them, three
proteins, including one member of the Pre-rRNA PROCESSING PROTEIN
(RRP5), one 30S ribosomal protein, and one COLD SHOCK DOMAIN CONTAINING
PROTEIN (CSP, named as MeCSP5), were found to be associated withCRIR1 RNA function, and the other candidate interactors were
mainly secondary metabolism‐related enzymes. CSP has been shown to
regulate RNA processing and function by participating in multiple
complexes to confer cold tolerance in Arabidopsis (Sasaki &
Imai, 2011). The cassava genome contains six CSPs (MeCSP1to 6 ) (Figure S2a-b), two of which (MeCSP1and MeCSP5 ) were regulated by cold stress according to the
RNA-seq data. Therefore, we verified possible direct interactions
between CRIR1 and MeCSP5. First, we predicted regions ofCRIR1 necessary for interaction with MeCSP5 using the catRAPID
database, and found that MeCSP5 was preferentially bound to the
N-terminal of CRIR1 (Figure 6c). Further, to check the
specificity of the interaction between CRIR1 and the MeCSP5
protein, full-length CRIR1 were separated into three truncated
forms 1-3, and were synthesized by in vitro transcription to
evaluate their ability to bind MeCSP5 protein. As presented in Figure
6d, MeCSP5 co-purified with transcripts of segments 1 and 2 but not
segment 3, demonstrating a sequence-specific binding activity of MeCSP5.
We also checked the interaction of CRIR1 with MeCSP5 using
trimolecular fluorescence complementation (TriFC) assay, a modified
version of bimolecular fluorescence complementation (BiFC) assay using
an MS2 system as described previously (Seo, Sun, Park, Huang, Yeh, Jung
& Chua, 2017). We observed that CRIR1 was strongly associated
with MeCSP5 in both the nucleus and cytoplasm, but the negative control
RNA was not (Figure 6e). Collectively, these results suggest an
association between CSP complexes and CRIR1 -mediated cold stress
tolerance. Considering that CSP proteins have been confirmed to possess
transcription anti-termination activity and have also been thought to
enhance translation at low temperatures by eliminating stabilized RNA
secondary structures (Sasaki & Imai, 2011). We thus compared the mRNA
and protein abundance in WT and transgenic plants. According to the
RNA-seq data, transcripts in WT and OE lines have a similar mean
abundance under normal conditions. However, low temperature strongly
reduced cellular mRNA levels in WT or OE lines (Figure 6f). Besides, we
found that the mean protein levels produced from OE lines were
significantly higher than that in WT plants under both normal and
cold-treated conditions based on the mean protein abundance (Figure 6g).
These data indicated that CRIR1 may potentially coordinatilate
with MeCSP5 may toincrease translational yield for coping with
cold stress.