Molecular Basis for Distinct, Antagonistic, Regulatory Functions of Ribosomal Protein Paralogs Rpl22 and Rpl22-Like1 (Like1) in Hematopoiesis

The prevailing view of ribosomal proteins (RP) is that they act to facilitate the core function of the ribosome in synthesizing proteins. However, emerging evidence suggests that some RP perform regulatory functions outside of the context of the ribosome ("extra-ribosomally"). Indeed, our laboratory demonstrated that ribosomal protein, Rpl22 and its paralog Rpl22l1 (Like1), are dispensable for ribosome assembly and translation, yet they perform critical regulatory functions in hematopoiesis that are mediated by binding selected RNA targets and controlling their translation. Despite their high degree of homology, Rpl22 and Like1 antagonistically control the emergence of hematopoietic stem cells (HSC) by regulating the translation of the key transcription factor, Smad1, with Rpl22 repressing its translation and Like1 acting to oppose that translation. Here, we seek to establish the molecular basis for how such highly homologous proteins perform distinct and opposing functions. Since both Rpl22 and Like1 share highly conserved RNA-binding domains in the central core of the proteins, we hypothesize that their unique regulatory functions are likely to reside in their termini. To systematically test this hypothesis, we employed a domain-swapping approach, creating a series of Rpl22:Like1 chimeric proteins to assess which changes would confer on Like1, the Rpl22 function of blocking HSC emergence. In doing so, we utilized zebrafish as a model organism. During embryonic zebrafish development, the enforced expression of Rpl22, but not Like1, represses Smad1 expression and blocks HSC) emergence. Interestingly, our analysis of the Rpl22:Like1 chimeras revealed that transfer of the N-terminal amino acids of Rpl22 to Like1 was sufficient to confer upon Like1 the Rpl22 function of blocking HSC emergence. Moreover, this Rpl22:Like1 N-terminal chimera also was able to restore the block in T lymphocyte development observed upon loss of Rpl22, indicating that Rpl22 function in another hematopoietic process, supporting T lymphocyte development, can be conferred upon Like1 by the N-terminal sequences of Rpl22. Together, these data suggest that the unique and opposing functions of Rpl22 and Like1 reside in their N-termini. How these newly identified N-terminal amino acid sequences confer distinct biological functions on these two paralogs is unclear at present, but may involve the association with distinct co-factors. This possibility is currently under investigation. This study demonstrates that relative minor changes in amino acid sequence can have profound effects on the functions of these RP paralogs, enabling them to play divergent regulatory roles in controlling hematopoiesis.