SUMMARY The Anaphase Promoting Complex/Cyclosome (APC/C) is an ubiquitin ligase and core component of the cell cycle oscillator. During G1-phase APC/C binds to its substrate receptor Cdh1 and APC/CCdh1 plays an important role in restricting S-phase entry and maintaining genome integrity. We describe a reciprocal feedback circuit between APC/C and a second ubiquitin ligase, the SCF (Skp1-Cul1-F box). We show that Cyclin F, a cell cycle regulated substrate receptor (F-box protein) for the SCF, is targeted for degradation by APC/C. Furthermore, we establish that Cdh1 is itself a substrate of SCFCyclin F. Cyclin F loss impairs Cdh1 degradation and delays S-phase entry, and this delay is reversed by simultaneous removal of Cdh1. These data indicate that the coordinated, temporal ordering of Cyclin F and Cdh1 degradation, organized in a double-negative feedback loop, represents a fundamental aspect of cell cycle control. This mutual antagonism could be a feature of other oscillating systems.

To better understand splicing regulation, we used a cell-based screen to identify ten diverse motifs that inhibit splicing from intron. Each motif was validated in another human cell type and gene context, and their presence correlated with in vivo splicing changes. All motifs exhibited exonic splicing enhancer or silencer activity, and grouping these motifs based on their distributions yielded clusters with distinct patterns of context-dependent activity. Candidate regulatory factors associated with each motif were identified, recovering 24 known and novel splicing regulators. Specific domains in selected factors were sufficient to confer ISS activity. Many factors bound multiple distinct motifs with similar affinity, and all motifs were recognized by multiple factors, revealing a complex, overlapping network of protein:RNA interactions. This arrangement enables individual cis-element to function differently in distinct cellular contexts depending on the spectrum of regulatory factors present.

Specific cleavage of RNAs is critical for in vitro manipulation of RNA and for in vivo gene silencing. Here we engineer artificial site-specific RNA endonucleases (ASREs) to function analogously to DNA restriction enzymes. We combine a general RNA cleavage domain with a series of Pumilio/FBF (PUF) domains that specifically recognize different 8-nt RNA sequences. The resulting ASREs specifically recognize RNA substrates and efficiently cleave near their binding sites. ASREs can be devised to recognize and cleave various RNA target sequences, providing a useful tool to manipulate RNAs in vitro. In addition, we generate designer ASREs to specifically silence an endogenous gene in E. coli, as well as a mitochondrial-encoded gene in human cells, suggesting that ASREs can serve as a gene silencing tool with designed specificity.

UBE3A is a HECT domain E3 ubiquitin ligase whose dysfunction is linked to autism, Angelman syndrome, and cancer. Recently, we characterized a de novo autism-linked UBE3A mutant (UBE3A T485A ) that disrupts phosphorylation control of UBE3A activity. Through quantitative proteomics and reporter assays, we found that the UBE3A T485A protein ubiquitinates multiple proteasome subunits, reduces proteasome subunit abundance and activity, stabilizes nuclear β-catenin, and stimulates canonical Wnt signaling more effectively than wild-type UBE3A. We also found that UBE3A T485A activates Wnt signaling to a greater extent in cells with low levels of ongoing Wnt signaling, suggesting that cells with low basal Wnt activity are particularly vulnerable to UBE3A T485A mutation. Ligase-dead UBE3A did not stimulate Wnt pathway activation. Overexpression of several proteasome subunits reversed the effect of UBE3A T485A on Wnt signaling. We also observed that subunits that interact with UBE3A and affect Wnt signaling are located along one side of the 19S regulatory particle, indicating a previously unrecognized spatial organization to the proteasome. Altogether, our findings indicate that UBE3A regulates Wnt signaling in a cell context-dependent manner and that an autism-linked mutation exacerbates these signaling effects. Our study has broad implications for human disorders associated with UBE3A gain or lossof-function, and suggest that dysfunctional UBE3A might affect additional proteins and pathways that are sensitive to proteasome activity.

Alternative splicing of pre-mRNA is a critical stage of gene regulation in response to environmental stimuli. Here we show that DAZAP1, an RNA binding protein involved in mammalian development and spermatogenesis, promotes inclusion of weak exons through specific recognition of diverse cis-elements. The C-terminal proline-rich domain of DAZAP1 interacts with and neutralizes general splicing inhibitors, and is sufficient to activate splicing when recruited to pre-mRNA. This domain is phosphorylated by the MEK/Erk pathway and this modification is essential for the splicing regulatory activity and the nuclear/cytoplasmic translocation of DAZAP1. Using mRNA-seq we identify endogenous splicing events regulated by DAZAP1, many of which are involved in maintaining cell growth. Knockdown or over-expression of DAZAP1 causes a cell proliferation defect. Taken together, these studies reveal a molecular mechanism that integrates splicing control into MEK/Erk regulated cell proliferation.