Elizabeth T. Abshire

Despite the importance of exon junction complexes (EJCs) to co- and post-transcriptional gene control, how and when the EJC core of EIF4A3, RBM8A, and MAGOH, and the peripheral factors of the alternative RNPS1-EJC and CASC3-EJC, assemble on spliceosomes remain unclear. Existing characterizations that order EJC assembly on spliceosomes offer different conclusions. Using immunoprecipitations (IPs) of EIF4A3, RNPS1, or CASC3 in the presence of RNase I followed by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) analyses of human HEK293T cells, we expand upon those splicing factors that associate with EJC constituents. Referencing these data against existing cryogenic electron microscopy (cryo-EM) spliceosome structures at different stages of pre-messenger RNA (pre-mRNA) splicing, coupled with data from a series of western blots of nuclear fractions before and after IP, we report that EIF4A3, RBM8A, MAGOH, and RNPS1 associate with assembled but inactive spliceosomes. This is followed by CASC3 joining to late activated spliceosomes. Our work bridges long-standing gaps in understanding EJC assembly on spliceosomes.

The exon junction complex (EJC) begins to assemble on the spliceosome, which deposits EJCs upstream of most exon–exon junctions during pre-messenger RNA (mRNA) splicing. EJCs acquire additional alternative modules that define heterogeneous EJCs during pre-mRNA processing to mRNA in the nucleus and after mRNA export into the cytoplasm. In this Review, we discuss the mechanisms of EJC formation, the many roles of the EJC in pre-mRNA and mRNA regulation and how these roles are influenced by EJC composition.

Post-transcriptional control of messenger RNA (mRNA) is an important layer of gene regulation that modulates mRNA decay, translation, and localization. Eukaryotic mRNA decay begins with the catalytic removal of the 3′ poly-adenosine tail by deadenylase enzymes. Multiple deadenylases have been identified in vertebrates and are known to have distinct biological roles; among these proteins is Nocturnin, which has been linked to circadian biology, adipogenesis, osteogenesis, and obesity. Multiple studies have investigated Nocturnin’s involvement in these processes; however, a full understanding of its molecular function remains elusive. Recent studies have provided new insights by identifying putative Nocturnin-regulated mRNAs in mice and by determining the structure and regulatory activities of human Nocturnin. This review seeks to integrate these new discoveries into our understanding of Nocturnin’s regulatory functions and highlight the important remaining unanswered questions surrounding its regulation, biochemical activities, protein partners, and target mRNAs.

The circadian protein Nocturnin (NOCT) belongs to the exonuclease, endonuclease and phosphatase superfamily and is most similar to the CCR4-class of deadenylases that degrade the poly-adenosine tails of mRNAs. NOCT-deficient mice are resistant to high-fat diet induced weight gain, and exhibit dysregulation of bone formation. However, the mechanisms by which NOCT regulates these processes remain to be determined. Here, we describe a pair of high-resolution crystal structures of the human NOCT catalytic domain. The active site of NOCT is highly conserved with other exoribonucleases, and when directed to a transcript in cells, NOCT can reduce translation and abundance of that mRNA in a manner dependent on key active site residues. In contrast to the related deadenylase CNOT6L, purified recombinant NOCT lacks in vitro ribonuclease activity, suggesting that unidentified factors are necessary for enzymatic activity. We also find the ability of NOCT to repress reporter mRNAs in cells depends upon the 3′ end of the mRNA, as reporters terminating with a 3′ MALAT1 structure cannot be repressed by NOCT. Together, these data demonstrate that NOCT is an exoribonuclease that can degrade mRNAs to inhibit protein expression, suggesting a molecular mechanism for its regulatory role in lipid metabolism and bone development.