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Hundreds of messenger RNAs (mRNAs) are transported into neurites to provide templates for the assembly of local protein networks. These networks enable a neuron to configure different cellular domains for specialized functions. According to current evidence, mRNAs are mostly transported in rather small packages of one to three copies, rarely containing different transcripts. This opens up fascinating logistic problems: how are hundreds of different mRNA cargoes sorted into distinct packages and how are they coupled to and released from motor proteins to produce the observed mRNA distributions? Are all mRNAs transported by the same transport machinery, or are there different adaptors or motors for different transcripts or classes of mRNAs? A variety of often indirect evidence exists for the involvement of proteins in mRNA localization, but relatively little is known about the essential activities required for the actual transport process. Here, we summarize the different types of available evidence for interactions that connect mammalian mRNAs to motor proteins to highlight at which point further research is needed to uncover critical missing links. We further argue that a combination of discovery approaches reporting direct interactions, in vitro reconstitution, and fast perturbations in cells is an ideal future strategy to unravel essential interactions and specific functions of proteins in mRNA transport processes. This study was supported by the Spanish Ministry of Economy and Competitiveness (MINECO) [BFU2017-85361-P], the Juan de la Cierva-Incorporación program [IJCI-2015-25994], the Human Frontiers in Science Program (HFSP) [RGY0083/2016], and the Ministerio de Ciencia, Innovación y Universidades and Fondo Social Europeo (FSE) [PRE2018-084501]. ECR was supported by an INPhINIT La Caixa PhD fellowship [LCF/BQ/DI19/11730046]. We further acknowledge the support of the Spanish Ministry of Economy and Competitiveness to the EMBL partnership, Centro de Excelencia Severo Ochoa [SEV-2012-0208] and [SEV-2015-0533], and the CERCA Program/Generalitat de Catalunya.

The complexity of the transcriptome is governed by the intricate interplay of transcription, RNA processing, translocation, and decay. In eukaryotes, the removal of the 5&rsquo, RNA cap is essential for the initiation of RNA degradation. In addition to the canonical 5&rsquo, N7-methyl guanosine cap in eukaryotes, the ubiquitous redox cofactor nicotinamide adenine dinucleotide (NAD) was identified as a new 5&rsquo, RNA cap structure in prokaryotic and eukaryotic organisms. So far, two classes of NAD-RNA decapping enzymes have been identified, namely Nudix enzymes that liberate nicotinamide mononucleotide (NMN) and DXO-enzymes that remove the entire NAD cap. Herein, we introduce 8-(furan-2-yl)-substituted NAD-capped-RNA (FurNAD-RNA) as a new research tool for the identification and characterization of novel NAD-RNA decapping enzymes. These compounds are found to be suitable for various enzymatic reactions that result in the release of a fluorescence quencher, either nicotinamide (NAM) or nicotinamide mononucleotide (NMN), from the RNA which causes a fluorescence turn-on. FurNAD-RNAs allow for real-time quantification of decapping activity, parallelization, high-throughput screening and identification of novel decapping enzymes in vitro. Using FurNAD-RNAs, we discovered that the eukaryotic glycohydrolase CD38 processes NAD-capped RNA in vitro into ADP-ribose-modified-RNA and nicotinamide and therefore might act as a decapping enzyme in vivo. The existence of multiple pathways suggests that the decapping of NAD-RNA is an important and regulated process in eukaryotes.