Insights into rRNA binding and lncRNAs in Brassicaceae

Abstract

Plants must quickly adapt to their surroundings due to their sessile nature. Their ability to adjust and thrive in diverse environmental conditions directly impacts their growth and development. This is especially critical for crop plants, where quantity and quality are vital to secure proper global nutrition. Plants have several ways to adapt or respond to external stimuli. One way is through the regulation of their proteome by maintaining the balance between protein synthesis and degradation. Another way is by emitting signaling molecules over short or long distances from affected areas to unaffected areas. The synthesis of proteins is controlled by ribosomes and their biogenesis is a multistep process that involves the synthesis and maturation of rRNAs with the aid of hundreds of ribosome biogenesis factors (RBFs). Although many RBFs have been identified in plants, a substantial amount remains to be thoroughly studied compared to other organisms. Thus, one of the aims of this work was to characterize the structure and function of the Nucleolar RNA Chaperone-like 1 (NURC1) RBF in A. thaliana. SEC-SAXS results showed that NURC1 is highly flexible and adopts an elongated shape in solution. SEC-MALLS confirmed that NURC1 was a monomer with a molecular weight of ~28 kDa and a Rh of 2.4 nm. The homogeneity of the sample was compromised by the increase of protein concentration as observed through DLS, native electrophoresis and batch SAXS measurements. RNA binding was determined by MST. NURC1 bound the ITS2 RNA of A. thaliana with a Kd of 228 ± 83 nM. Furthermore, NURC1 displayed an RNA chaperone-like activity by FRET. The flexible termini of NURC1 seemed to be essential for structural stability and RNA recognition and/or biding since their truncation led to either increase of the sample polydispersity, decreased the binding affinity towards the ITS2 or reduced the RNA chaperone-like activity. Long-distance communication is achieved through the phloem. Several classes of RNA have been identified in the phloem sap, including long non-coding RNAs (lncRNAs), which can potentially work as signals. The crop plant B. napus allows to readily collect fair amounts of phloem sap by exudation. Thus far, the population of lncRNAs of B. napus phloem sap remains to be explored. Hence, the second aim of this thesis involved the identification and characterization of lncRNAs in the phloem sap of B. napus by Illumina sequencing. For comparison, leaf samples were also analyzed. Over 3000 putative lncRNAs were identified in phloem and leaf samples. No structural differences were found between leaf and phloem lncRNAs. Differential expression and functional analysis (GO terms) were also performed. Parent genes associated with various molecular functions or pathways were identified, likely due to the diversity of processes that lncRNAs are involved.