4.4 CRIR1 functions related to MeCSP5 in cassava
MeCSPs are nucleic acid-binding proteins that contain both cold shock domain (CSD) and DNA-binding domains, which have been widely found in bacteria, animals, and plants. CSPs are important during cold shock response. Besides, the functions of CSPs were first described in Escherichia coli (Goldstein, Pollitt & Inouye, 1990). During cold stress, cell membrane fluidity and enzyme activity decrease, and transcription and translation efficiency is reduced to stabilize nucleic acid secondary structures. CSPs function as RNA chaperones and counteract these harmful effects by destabilizing secondary structures in target RNA at low temperatures, thus facilitating the translation (Keto-Timonen, Hietala, Palonen, Hakakorpi, Lindstrom & Korkeala, 2016). Arabidopsis AtCSP3 shares an RNA chaperone function with Escherichia coli  cold shock proteins and positively regulates freezing tolerance through a non-CBFpathway (Kim et al., 2009). In this study, CRIR1 was showed to be able to interact with MeCSP5, a homology protein of ArabidopsisAtCSP2 with 50% identity.. In addition, MeCSP5 protein exhibited a similar cellular distribution pattern with CRIR1 transcripts (Figure S2c). Although cold stress dramatically stimulated theMeCSP5 gene expression, its transcript level was not significantly altered in the CRIR1 -overexpressors (Figure S2d), suggesting that CRIR1 could regulate MeCSP5 at protein level. In Arabidopsi s, AtCSP3 protein has been shown to interact with a range of proteins in the nucleolus, nucleoplasm, and cytoplasm, including ribosomal proteins, Gar1 RNA-binding protein, and mRNA decapping protein. The results suggested that AtCSP3 participated in multiple complexes and possibly regulated RNA processing and translation (Kim, Sonoda, Sasaki, Kaminaka & Imai, 2013). One ribosomal protein was found to interact with CRIR1 , indicating the possible involvement of CRIR1 in ribosome biogenesis and protein translation. Therefore, it will be interesting to determine if the complex that forms with CRIR1 , MeCSP5 and other proteins regulates cold stress tolerance in cassava by controlling the RNA unwinding activity or facilitating mRNA translation.
Taken together, in this study, a total of 3 004 full-length lncRNAs in cassava were identified, and 316 of them were further characterized as cold-related lncRNAs. Functional analysis shows that CRIR1 can confer cold stress tolerance and participate in MeCSP complexes to enhance translation efficiency at low temperatures. The research has provided a valuable source for studying full-length lncRNAs in cassava and their corresponding candidate genes. CRIR1 , as a case in the study, will provide applicable genetic resource for breeding cold tolerant crops without yield penalty.