Affinity proteomic dissection of the human nuclear cap-binding complex interactome

Abstract

A 5', 7-methylguanosine cap is a quintessential feature of RNA polymerase II-transcribed RNAs, and a textbook aspect of co-transcriptional RNA processing. The cap is bound by the cap-binding complex (CBC), canonically consisting of nuclear cap-binding proteins 1 and 2 (NCBP1/2). The CBC has come under renewed investigative interest in recent years due to its participation in RNA-fate decisions via interactions with RNA productive factors as well as with adapters of the degradative RNA exosomeincluding the proteins SRRT (a.k.a. ARS2) and ZC3H18, and macromolecular assemblies such as the nuclear exosome targeting (NEXT) complex and the poly(A) exosome targeting (PAXT) connection. A novel cap-binding protein, NCBP3, was recently proposed to form an alternative, non-canonical CBC together with NCBP1, and to interact with the canonical CBC along with the protein SRRT. The theme of post-transcriptional RNA fate, and how it relates to co-transcriptional ribonucleoprotein assembly is abundant with complicated, ambiguous, and likely incomplete models. In an effort to clarify the compositions of NCBP1-, 2-, and 3-related macromolecular assemblies, including their intersections and differences, we have applied an affinity capture-based interactome screening approach, where the experimental design and data processing have been modified and updated to identify interactome differences between targets under a range of experimental conditions, in the context of label-free quantitative mass spectrometry. This study generated a comprehensive view of NCBP-protein interactions in the ribonucleoprotein context and demonstrates the potential of our approach to benefit the interpretation of complex biological pathways.

NCBP3 positively impacts mRNA biogenesis

“…To obtain comprehensive interaction profiles, we applied an optimized IP screen, where multiple extraction conditions were used to favor a broad range of protein interactions ( 36 ). Details of this IP screen are described elsewhere ( 37 ). After a preliminary screen with 24 different extraction conditions, final IPs were conducted in triplicate or quadruplicate using six different conditions for NCBP1 and NCBP2, and four different conditions for NCBP3 and the control IP (see Supplemental Table S1 for solution details).…”

“…In contrast, the most predominant interactors of NCBP3 were components of the EJC and TREX complexes, among which EJC core components EIF4A3, RBM8A and MAGOH were most abundantly enriched across all IP conditions ( Supplemental Figure S2 ). To better compare interaction differences between each of the NCBP proteins, we summarized protein groups/complexes that abundantly interact with NCBP3 or NCBP1 and NCBP2 in a heatmap (Figure 2A , for extensive complex enrichment comparison ( 37 )). PHAX was exclusively, and ARS2 was primarily, enriched in NCBP1 and NCBP2, but not in NCBP3, IPs, which was consistent with previous studies ( 16 , 32 ).…”

“…The interaction screening was performed essentially as described in ( 36 , 37 ). Briefly, three or four replicate IPs were conducted, for each of the extraction conditions.…”

“…Details of MS sample processing is described elsewhere ( 37 ). Briefly, samples were reduced, alkylated and in-gel trypsin digested.…”

Cytoplasmic Switch of ARS2 Isoforms Promotes Nonsense-Mediated mRNA Decay and Arsenic Sensitivity

“…We know from previous work that ARS2n interacts with all four core components of the EJC: CASC3, MAGOH, RBM8A and EIF4A3, the EJC periferal factors: UPF3B, RNPS1, PNN and ACINUS as well as UPF1 and the phosphatases PPP2R1A and PPP2R2A (Fig. 4E) 5,19,20,43–46 . Additionally, the CBC complex (NCBP1/2) has been shown to stabilize the interaction between UPF1-ERF1/ERF3 and the EJC, promoting NMD 9,18 .…”