The interactions of the RNA recognition motif protein Sex -Lethal with its substrate, the single-stranded transformer pre -mRNA

The RNA-binding protein Sex-Lethal (SXL) has high affinity for poly-uridine sequences found near the regulated 3' splice site of the Transformer pre-mRNA (tra). SXL contains two RNA Recognition Motifs (RRM) connected by a small linker. Each motif has a βαββαβ pattern with two RNP conserved sequences containing amino acids that are responsible for interactions with uridine-rich tracts in RNA. We have examined the SXL/tra stoichiometry and the possibility that SXL binds tra as a dimer. We have attempted to identify the presence of the dimer and of an aggregate complex, binding tra non-specifically. The most probable SXL/tra stoichiometry is 2:1 and the data indicates a dimer may be the prevailing complex with the aggregate forming at high protein concentrations, in the micromolar range. We have investigated the key interactions affecting the affinity of the complex between SXL and tra, hydrogen bonding and stacking interactions, to determine which interactions are most important or if a combination of both is needed for affinity. We also have sought to ascertain which interactions govern the SXL/tra recognition process. We have mutated five amino acids; in RRM1 Y131 and in RRM2 Y214 to phenylalanine and alanine, in RRM1 F170 and in RRM2 F256 to alanine, in the linker R202 to alanine. We also used single domains RRM1 and RRM2. F170A, F256A, Y131A have the greatest decrease in affinity, indicating that stacking interactions are the primary interactions needed for affinity, not hydrogen bonding. RRM1 and RRM2 did not bind tra singly, emphasizing that both domains must be present to bind tra, and the R202A mutation confirmed the importance of hydrogen bonding between the linker, tra, and RRM2 for affinity. We mutated the RNA sequence at position 10, 11, and 16 with 2,4-difluorotoluyluridine and found the greatest decrease in affinity at position 10, corresponding to hydrogen bonding as the most prevalent interactions for U10. However, this result cannot be generalized. We believe that recognition of tra by SXL may be modulated differently by each domain RNP sequence and that hydrophobic interactions may be the primary mode of recognition.