Post-transcriptional Processing of mRNA in Neurons: The Vestiges of the RNA World Drive Transcriptome Diversity

Abstract

Neurons are morphologically complex cells that rely on the compartmentalization of protein expression to develop and maintain their extraordinary cytoarchitecture. This formidable task is achieved, at least in part, by targeting mRNA to subcellular compartments where they are rapidly translated. mRNA transcripts are the conveyor of genetic information from DNA to the translational machinery, however, they are also endowed with additional functions linked to both the coding sequence (open reading frame, or ORF) and the flanking 5' and 3' untranslated regions (UTRs), that may harbor coding-independent functions. In this review, we will highlight recent evidences supporting new coding-dependent and -independent functions of mRNA and discuss how nuclear and cytoplasmic post-transcriptional modifications of mRNA contribute to localization and translation in mammalian cells with specific emphasis on neurons. We also describe recently developed techniques that can be employed to study RNA dynamics at subcellular level in eukaryotic cells in developing and regenerating neurons.

Keywords: 3′UTR; RNA isoforms; RNA localization; RNA metabolism; RNA processing; neuron; translation.

FIGURE 1

The RNA world postulates that the origin of life coincides with the appearance of the primordial ribozyme, which conjugated coding functions in the nucleotide sequence with enzymatic non-coding functions. This molecule generated the first short polypeptides and through evolution, gave rise to the multifaceted modern RNAs. mRNA, messenger RNA; tRNA, transfer RNA; miRNA, microRNA; lncRNA, long non-coding RNA.

FIGURE 2

(A) Schematic representation of a mRNA transcript. Isoforms can be generated by alternative transcription start site (TTS) choice (B), exon splicing or intron retention (C) or alternative transcriptional termination site (TSS) and polyadenylation site (PAS) (D). The combination of two or more mechanisms for the same gene further increases the diversity and number of isoform expression. P, proximal PAS; D, distal PAS.

FIGURE 3

(A) 3′UTR remodeling. Transcripts expressing a long 3′UTR can undergo post-transcriptional cleavage and remodeling in response to extrinsic stimuli. The cleavage is mediated by the endonuclease Argonaute2 (Ago2), the helicase UPF1 and the recruitment of the complex to the transcripts involves the RNA binding protein HuD. (B) Circular RNAs (circRNAs) are generated by a mechanism of backsplicing that can give rise to transcripts expressing exons, introns or both. (C) mRNA transcripts may have multiple functions. (1) In some cases, the same gene encodes coding-dependent and coding-independent isoforms. (2) The Arc transcript is translated into a protein that interacts with Arc mRNA to form a virus-like structure. (3) The same transcripts may be translated in some cells and have coding-independent functions in others, interacting with membrane receptors, for example.

FIGURE 4

Methods to study mRNA isoform expression and metabolism. (A) Sequencing-based methods. Microdissection of specific cell populations (left) or Fluorescent in situ RNA Sequencing (right) allows spatially resolved transcriptomics. (B) Single molecule FISH (smFISH) and related techniques. Single labeled multiple probes (top left), padlock probes and rolling circle amplification (top right), branched DNA probes (bottom left) or aptamers and related fluorogens, such as Spinach (bottom right) have lowered the threshold of mRNA detection to single molecule. (C) Orthogonal systems of RNA stem-loops are engineered in the mRNA of interest and binding proteins are conjugated to fluorescent proteins. The most popular methods to study mRNA transport, local translation and degradation include MS2 stem-loops and MCP-GFP (top) and tandem array peptides engineered in the mRNA of interest and fluorescent single-chain variable fragment antibody (SunTag system, bottom).