Molecular Basis for the Recognition of the Regulatory Stem-loop Structures in Eukaryotic Messenger RNAs

Apart from carrying genetic information, RNAs also act as effectors of cellular processes through folding into intricate secondary and tertiary structures. The ubiquitous RNA structures in eukaryotic mRNAs, in collaboration with specific RNA-binding proteins, control many aspects of the post-transcriptional regulation of gene expression. However, the molecular bases for the recognition of these mRNA structures by their protein partners remain poorly understood due to the lack of structural information. This dissertation presents our structural studies on two protein-RNA complexes that both include regulatory mRNA stem-loop structures. We first describe the crystal structure of a ternary complex including the highly conserved human histone mRNA stem-loop (SL), the stem-loop binding protein (SLBP) and the 3′ to 5′ exonuclease 3′hExo. This structure identifies a single sequence-specific interaction between the SL and SLBP, and the mostly shape-dependent RNA-recognition mode by both proteins. In addition to explaining the large body of biochemical and biophysical data on this complex accumulated over the last two decades, we also for the first time elucidate the induced-fit mechanism underlying the cooperativity between SLBP and 3′hExo. We next shift our focus to a class of less conserved mRNA stem-loop structures named constitutive decay elements (CDE). The RNA-binding ROQ domain of Roquin recognizes the various CDEs and mediates the decay of CDE-containing mRNAs, which predominantly encode proteins responsible for inflammation and autoimmunity. Structural and biochemical studies of the ROQ domain in complex with two different CDE RNAs unexpectedly reveal two distinct RNA binding sites on this protein, one recognizing CDE stem-loops and the other binding to double-stranded RNAs. The stuctures are also in agreement with the versatility of Roquin and have opened up new avenues to investigating its functions in modulating the stability of target mRNAs.