Coexpression of proteins in response to pathway-inducing signals is the founding paradigm of gene regulation. However, it remains unexplored whether the relative abundance of co-regulated proteins requires precise tuning. Here, we present large-scale analyses of protein stoichiometry and corresponding regulatory strategies for 21 pathways and 67-224 operons in divergent bacteria separated by 0.6-2 billion years. Using end-enriched RNA-sequencing (Rend-seq) with single-nucleotide resolution, we found that many bacterial gene clusters encoding conserved pathways have undergone massive divergence in transcript abundance and architectures via remodeling of internal promoters and terminators. Remarkably, these evolutionary changes are compensated post-transcriptionally to maintain preferred stoichiometry of protein synthesis rates. Even more strikingly, in eukaryotic budding yeast, functionally analogous proteins that arose independently from bacterial counterparts also evolved to convergent in-pathway expression. The broad requirement for exact protein stoichiometries despite regulatory divergence provides an unexpected principle for building biological pathways both in nature and for synthetic activities.

SUMMARY Constituents of multi-protein complexes are required at well-defined levels relative to each other. However, it remains unknown whether eukaryotic cells typically produce precise amounts of subunits, or instead rely on degradation to mitigate imprecise production. Here we quantified the production rates of multi-protein complexes in single- and multi-cellular eukaryotes using ribosome profiling. By resolving read-mapping ambiguities which occur for a large fraction of ribosome footprints and distort quantitation accuracy in eukaryotes, we found that obligate components of multi-protein complexes are produced in proportion to their stoichiometry, indicating that their abundances are already precisely tuned at the synthesis level. By systematically interrogating the impact of gene dosage variations in budding yeast, we found a general lack of negative feedback regulation protecting the normally precise rates of subunit synthesis. These results reveal a core principle of proteome homeostasis and highlight the evolution towards quantitative control at every step in the central dogma.

No abstract

The MUSASHI (MSI) family of RNA binding proteins (MSI1 and MSI2) contribute to a wide spectrum of cancers including acute myeloid leukemia. We find that the small molecule Ro 08–2750 (Ro) binds directly and selectively to MSI2 and competes for its RNA binding in biochemical assays. Ro treatment in mouse and human myeloid leukemia cells results in an increase in differentiation and apoptosis, inhibition of known MSI-targets, and a shared global gene expression signature similar to shRNA depletion of MSI2. Ro demonstrates in vivo inhibition of c-MYC and reduces disease burden in a murine AML leukemia model. Thus, we identify a small molecule that targets MSI’s oncogenic activity. Our study provides a framework for targeting RNA binding proteins in cancer.

How do cells maintain relative proportions of protein complex components? Advances in quantitative, genome-wide measurements have begun to shed light onto the roles of protein synthesis and degradation in establishing the precise proportions in living cells: on the one hand, ribosome profiling studies indicate that proteins are already produced in the correct relative proportions. On the other hand, proteomic studies found that many complexes contain subunits that are made in excess and subsequently degraded. Here, we discuss these seemingly contradictory findings, emerging principles, and remaining open questions. We conclude that establishing precise protein levels involves both coordinated synthesis and post-translational fine-tuning via protein degradation.

The identity of the RNA binding proteins (RBPs) that govern cancer stem cell remains poorly characterized. The MSI2 RBP is a central regulator of translation of cancer stem cell programs. Through proteomics analysis of the MSI2 interacting RBP network and functional shRNA screening, we identified 24 genes required for in vivo leukemia and SYNCRIP was the most differentially required gene between normal and myeloid leukemia cells. SYNCRIP depletion increased apoptosis and differentiation while delaying leukemogenesis. Gene expression profiling of SYNCRIP depleted cells demonstrated a loss of the MLL and HOXA9 leukemia stem cell gene associated program. SYNCRIP and MSI2 interact indirectly though shared mRNA targets. SYNCRIP maintains HOXA9 translation and MSI2 or HOXA9 overexpression rescued the effects of SYNCRIP depletion. We validated SYNCRIP as a novel RBP that controls the myeloid leukemia stem cell program and propose that targeting these functional complexes might provide a novel therapeutic strategy in leukemia.

Summary Leukemias exhibit a dysregulated developmental program mediated through both genetic and epigenetic mechanisms. Although IKZF2 is expressed in hematopoietic stem cells (HSCs), we found that it is dispensable for mouse and human HSC function. In contrast to its role as a tumor suppressor in hypodiploid B-Acute Lymphoblastic Leukemia, we find that IKZF2 is required for myeloid leukemia. IKZF2 is highly expressed in leukemic stem cells (LSCs) and its deficiency results in defective LSC function. IKZF2 depletion in AML cells reduced colony formation, increased differentiation and apoptosis, and delayed leukemogenesis. Gene expression, chromatin accessibility and direct IKZF2 binding in MLL-AF9 LSCs demonstrate that IKZF2 regulates a HOXA9 self-renewal gene expression program and inhibits a C/EBP-driven differentiation program. Ectopic HOXA9 expression and CEBPE depletion rescued the effects of IKZF2 depletion. Thus, our study shows that IKZF2 regulates the AML LSC program and provides a rationale to therapeutically target IKZF2 in myeloid leukemia.

SUMMARY 37The MUSASHI family of RNA binding proteins (MSI1 and MSI2)