Most mRNAs are transported to neurites within RNA granules, which are dynamic, membrane-less cellular structures that contain mRNA molecules and various proteins (Dalla Costa et al., 2021). Interestingly, recent imaging experiments showed that the dynamics of endogenous RNA granules correlate with new branch emergence and branch stabilization (Wong et al., 2017), indicating that localized mRNAs play a role in the formation and stabilization of neural connections. Traditionally, protein synthesis was thought to occur exclusively in the soma cytoplasm. However, it has become increasingly evident that local mRNA translation can occur, and is widespread, at specific subcellular locations within neurons. During local protein synthesis, mRNA molecules are translated into proteins near the site they are required. Such local protein synthesis sites range from axonal and dendritic branch points to developing and mature pre- and post-synaptic compartments, as well as near cellular organelles (Figure 2). It is thought that up to half of the proteome in neurites has local protein synthesis being the predominant source (Zappulo et al., 2017; Glock et al., 2021).

Within axons, dendrites and synaptic compartments, an increasing number of studies have begun to reveal the importance of associations between organelles and ribonucleoprotein (RNP) complexes for local translation (Pushpalatha and Besse, 2019; Vargas et al., 2022). Indeed, RNA-bearing Rab7a late endosomes were found to pause on mitochondria along RGC axons, facilitating translation of mRNAs encoding mitochondrial proteins (Cioni et al., 2019). This mode of local translation was shown to be important for mitochondrial function and axonal viability. In another study, tethering of certain transcripts to axonal mitochondria has been shown to be important for their translation to maintain the mitophagy pathway (Harbauer et al., 2022). In a late-endosomme independent manner, PINK1 mRNAs require tethering to the mitochondrial outer membrane by Synaptojanin2 (SYNJ2), for their transport and translation (Harbauer et al., 2022). Intriguingly, translation of the PINK1 mitochondrial targeting sequence was also required for such transport, suggesting a local translated peptide was essential for the localisation of its own transcript to neurites. Other studies have identified important roles for the endoplasmic reticulum (ER) in local translation. It was recently shown that ribosomes associate with ER upon activation of local translation in motor axonal growth cones following their stimulation with brain-derived neurotrophic factor (BDNF) (Deng et al., 2021). It is likely that these ribosomes translate membrane and secreted proteins, classically translated at the rough ER, which was not known to occupy axons prior. Another study also showed ribosomes contacting ER tubules in a translation-dependent manner, in a process facilitated by the axonal ribosome/mRNA receptor P180/RRBP1 (Koppers et al., 2022). Future studies will reveal the contribution of other organelles to local translation in neurites, and identify which mRNAs require specific organelles for the process.

Global pools of locally translated mRNAs in neurites have been revealed by studies using novel ribosome capturing and RNA sequencing techniques. One such technique was the development of axon-TRAP-RiboTag (Shigeoka et al., 2016), utilizing a mouse line harboring a modified allele of the ribosomal Rpl22 protein fused to a HA tag (Rpl22-HA), induced by the action of a Cre recombinase (Sanz et al., 2009). Using a RGC-specific Cre line, full-length mRNAs pulled-down with HA-tagged ribosomes revealed the local translatome within RGC axons at multiple stages (Shigeoka et al., 2016). This identified a dominance of mRNAs encoding proteins involved in vesicle-mediated transport and calcium-mediated signaling. Ribosome immunoprecipitation approaches have also been used to identify locally translated transcripts in dendrites isolated from adult mouse hippocampus, revealing a dominance of mRNAs encoding translation and cytoskeletal proteins (Ainsley et al., 2014). Transcripts encoding nuclear proteins, including histones, were also observed, as in axons (see above section on mRNA diversity).

An alternative method for determining which proteins are actively translated locally is ribosome footprinting/profiling (Ingolia et al., 2012). Also known as Ribo-Seq (ribosome sequencing) or ART-Seq (active mRNA translation sequencing), it provides a snapshot, revealing RNA fragments/“footprints” protected by ribosomes caught during active translation. To identify and quantify the transcriptome and translatome in cell bodies (somata) as well as dendrites and axons, a recent study performed simultaneous RNA-Seq and Ribo-Seq from micro-dissected hippocampal rodent brain slices (Glock et al., 2021). The study led to the identification of more than 800 mRNAs whose dominant source of translation is the neuropil, suggesting that many axonal/dendritic and synaptic proteins arise mostly from local translation (Glock et al., 2021). But how do these localized mRNAs undergo local protein synthesis? During translation in the soma, multiple ribosomes can occupy an individual mRNA (a complex called a polysome), resulting in the simultaneous generation of multiple copies of the encoded protein. A recent study showed that monosomes (single ribosomes), as opposed to polysomes, are the predominant ribosome population in neuronal processes (Biever et al., 2020). Indeed, measuring ribosome density on transcripts in synaptic neuropil, revealed monosomes predominantly elongate key synaptic transcripts in both dendritic and axon terminals (Biever et al., 2020). One possible explanation for the difference between somatic and local mRNA translation that could explain the high abundance of monosomes in the neuropil, is the production of a more diverse set of proteins from a limited pool of available ribosomes found at synapses (Ostroff et al., 2017).

Local protein synthesis is a highly regulated process, with most local transcripts not translated by default. Some of the most abundant transcripts in neurites seemingly do not associate with ribosomes (Table 1; von Kügelgen and Chekulaeva, 2020). Studies comparing the mRNA species constituting local transcriptomes and those associating with ribosomes, reveal that only specialized subsets of transcripts become translated, in a spatio-temporal fashion. Furthermore, ribosomal footprinting data from synaptoneurosomes reported that mRNAs undergoing translation were associated with different ontologies (mitochondrial and extracellular matrix and exosome proteins) to those generally dominant (see above in mRNA diversity section) (Simbriger et al., 2020).

How dynamic is the local translatome? Certain mRNAs encoding regulators of protein and energy homeostasis, and those associated with vesicle-mediated transport and calcium-mediated signaling are translated regardless of developmental stage (Shigeoka et al., 2016). Other mRNAs are dynamically regulated during development and maturation, suggesting that local translation plays an important role in the homeostasis of neurites. The translatome of younger axons was enriched for GO terms such as neuron projection morphogenesis (Shigeoka et al., 2016). Contrastingly, the adult axonal translatome was found to have strong links to axon survival, neurodegenerative disease, and neurotransmission, with key components of the trans-SNARE complex, which mediates neurotransmitter exocytosis, being highly translated in mature axons (Shigeoka et al., 2016). The findings indicating that axonal mRNA translation persists in adult CNS axons were intriguing because it has been controversial whether mature CNS axon terminals can synthesize proteins at all, partly because of early studies detecting few or no ribosomes in mature axons (Koenig et al., 2000). Therefore, these findings showed conclusively a unique adult local translatome is present in mature axons, whose main role is likely to be the regulation of synapse function. In contrast, local translation of transcripts involved in axonal and dendritic elongation, branching, pruning, synaptogenesis, and synaptic transmission occurs developmentally (Shigeoka et al., 2016; Biever et al., 2020), indicating the process has an equally crucial role in regulating neuronal connectivity and synaptic plasticity. Indeed, local translation is thought to enable neuronal cells to respond to signals from the environment. For instance, extracellular cues (e.g., Netrin-1, BDNF, Sema3A) were found to differentially influence axonal synthesis of multiple proteins in a cue-specific and temporally dynamic manner. Interestingly, the synthesis of proteasomal subunits (α and β type), some ribosomal proteins, histones, and methyltransferases is differentially regulated in response to such cues (Cagnetta et al., 2018). The significance of local mRNA translation in neurite growth is supported by functional experiments in Xenopus laevis RGC axons (Wong et al., 2017). Here, local protein synthesis was found to be essential for proper axon arbor formation in vivo, as inhibition of local translation or knockdown of local β-actin synthesis caused a marked reduction in axon branching dynamics and arbor complexity (Wong et al., 2017).

Local protein synthesis is also required for synaptic plasticity. At synapses, local protein synthesis was found to be differentially recruited to drive compartment-specific phenotypes that may underlie different forms of plasticity (Hafner et al., 2019). Evidence for a role of local translation in synaptic plasticity comes from a study utilizing dissociated rat hippocampal neuron cultures. During basal synaptic transmission, the amount of locally synthesized proteins detected at a synapse was correlated with its level of ongoing spontaneous activity. Plasticity induced by single-spine stimulations or by a global activity manipulation resulted in a significant increase in local protein synthesis (Sun et al., 2021). Similarly, depolarization of primary cortical neurons caused rapid reprogramming of dendritic protein expression (Hacisuleyman et al., 2024).

Many locally translated transcripts interact with RNA binding protein (RBPs) through sequences found within the non-coding untranslated regions (UTRs) (Andreassi et al., 2018). Such interactions have been shown to regulate local translation. A well-known negative regulator is Fragile X Messenger Ribonucleoprotein (FMRP), which has been shown to interact with the coding region and 3′ UTR of many mRNA transcripts encoding pre- and post-synaptic proteins, many of which were found to be linked to autism (Darnell et al., 2011; Ouwenga et al., 2017). These RNA-protein interactions repress the local translation of transcripts, with dendritic (Hale et al., 2021) and axonal (Jung et al., 2023) FMRP target mRNAs showing increased ribosome association in Fmr1 knockout mice. RBFOX1, which regulates the splicing of many exons in neurons, binds to the 3′ UTR of cytoplasmic mRNA targets involved in cortical development and autism to increase their stability and local translation (Lee J. A. et al., 2016). Another positive regulator of local protein synthesis is PTBP2, which was shown to interact with the 3′ UTR of Hnrnpr mRNA, mediating the association of Hnrnpr with ribosomes in a translation factor eIF5A2-dependent manner (Salehi et al., 2023). Indeed, local synthesis of hnRNPR protein is strongly reduced when PTBP2 is depleted, leading to defective motor axon growth (Salehi et al., 2023).

It has been hypothesized that longer UTR sequences may permit a greater number of binding motifs for post-transcriptional regulation, including increased local protein synthesis (Andreassi and Riccio, 2009). Thus, an additional mechanism for regulating local protein synthesis could be through alternative splicing, such as the production of transcripts with alternative last exons (ALEs), and thus distinct 3′ UTRs. Indeed, transcripts with ALEs are disproportionately found in neurites (Taliaferro et al., 2016) undergoing local mRNA translation (Ouwenga et al., 2017). Moreover, cis-regulatory elements generated by alternative splicing at 5′ and 3′ UTRs have been shown to promote axonal mRNA translation (Shigeoka et al., 2016). Therefore, alternative splicing at the UTRs could influence the ability of transcripts to be locally translated. Control of mRNA translation in neuronal subcellular compartments is discussed in more detail elsewhere (Cagnetta et al., 2023).

3′ UTRs are involved in many aspects of mRNA metabolism, including intracellular localisation and translation. Surprisingly, global mapping of 3′ end regions indicated that ∼75% of mammalian genes contain more than one polyadenylation (poly(A)) site (PAS), giving rise to multiple 3′ UTRs (Proudfoot, 2011; Tian and Manley, 2013; Gruber and Zavolan, 2019). There is remarkable variation in PAS and 3′ UTR length between tissues, with neurons characterized by significantly longer 3′ UTRs (Miura et al., 2013). During neuronal development, many genes are subjected to 3′ UTR and/or poly(A) lengthening (Miura et al., 2013; Kiltschewskij et al., 2023), suggesting this constitutes an important mechanism of post-transcriptional mRNA regulation associated with neuronal differentiation. The process is thought to be a mechanism that serves unique post-transcriptional regulatory needs of transcripts in neurons e.g., transcript localisation, stabilization, and local protein synthesis regulation (Miura et al., 2014).

Although 3′ end cleavage and polyadenylation predominantly occur in the soma, evidence for local processing of alternative 3′ UTR isoforms has also been observed in axons and dendrites (Figure 2). Within neurites, many local mRNA transcripts have long 3′ UTRs and have significantly longer half-lives than somata-enriched isoforms (see mRNA stability and degradation section below) (Tushev et al., 2018). Interestingly, these 3′ UTR isoforms can be significantly altered by neuronal activity, with elevated activity resulting in significant shortening of neuropil-localized 3′ UTR isoforms (Tushev et al., 2018). Although most 3′ UTR plasticity was found to be transcription-dependent, evidence for transcription-independent changes was also reported (Tushev et al., 2018), hypothesized to arise from altered stability, trafficking of 3′ UTR isoforms between soma and neuropil, or local remodeling of 3′ UTRs by shortening or lengthening. Direct evidence for local cleavage and polyadenylation comes from work on rat sympathetic neurons showing that axons and cell bodies express distinct pools of 3′ UTR isoforms (Andreassi et al., 2021). Axon-specific short 3′ UTR isoforms of IMPA1, Maoa, and Sms are generated through a process of 3′ UTR cleavage and polyadenylation in axons. This local processing generates translatable isoforms necessary for maintaining the integrity of sympathetic neuron axons (Andreassi et al., 2021). Local cleavage and polyadenylation are further supported by a recent study showing that exposure of sympathetic neurons to Nerve Growth Factor (NGF) or Neurotrophin 3 (NT-3) induces the localization of distinct 3′ UTR isoforms to axons, including short 3′ UTR isoforms found exclusively in axons (Luisier et al., 2023). These observations support a model whereby long 3′ UTR isoforms associate with RBP complexes in the nucleus and, upon reaching the axons, are remodeled locally into shorter isoforms.

A key factor controlling non-nuclear polyadenylation is cytoplasmic polyadenylation binding protein (CPEB), an RBP with strong association for the cis-acting cytoplasmic polyadenylation element (CPE) residing in 3′ UTRs of target mRNAs. CPEB regulates poly(A) tail length by interacting with deadenylating enzymes as well as noncanonical poly(A) polymerases. Many of the components of the cytoplasmic polyadenylation machinery have been found at post-synaptic sites of hippocampal neurons, including CPEB, the scaffold protein Symplekin, the deadenylase poly(A) ribonuclease (PARN), the noncanonical poly(A) polymerase germ line defective 2 (Gld2), and CPEB-interacting factor neuroguidin (Ngd) (Jung et al., 2006; Udagawa et al., 2012; Swanger et al., 2013). The decision whether CPEB binds a deadenylating enzyme (e.g., PARN) favoring short poly(A) tails and translational dormancy, or noncanonical poly(A) polymerases (e.g., Gld2) favoring elongated poly(A) tails and translation, depends on its phosphorylation (Barnard et al., 2004, 2005). Generally, synaptic stimulation promotes phosphorylation, which in turn stimulates poly(A) tail lengthening and local translation (Ivshina et al., 2014).

The cytoplasmic polyadenylation machinery locally acts to bidirectionally regulate mRNA-specific translation and plasticity at hippocampal synapses in response to synaptic transmission, with the poly(A) tail of 102 mRNAs shortened following depletion of Gld2 (Udagawa et al., 2012). One such local transcript is NR2A (or GluN2A) mRNA, encoding an NMDA receptor subunit, which contains CPEs in its 3′ UTR, has a short poly(A) tail and is translated inefficiently (Udagawa et al., 2012). NR2A RNA is bound by CPEB, which in turn is associated with PARN, Gld2, Symplekin, and Ngd. However, because Ngd is also bound to the cap binding factor, eIF4E, translation is blocked at initiation. NMDA receptor activation was found to promote phosphorylation of CPEB, expulsion of PARN from the RNP complex, and Gld2-catalyzed poly(A) lengthening of NR2A mRNA (Udagawa et al., 2012). This local polyadenylation is thought to displace Ngd from eIF4E, the binding of eIF4G to eIF4E, resulting in enhanced translation of NR2A mRNA and membrane insertion of NMDA receptors in dendrites (Swanger et al., 2013). These findings indicate that local polyadenylation has an important role in the activity-dependent synthesis, and NMDA receptor surface expression during synaptic plasticity. Indeed, depletion of CPEB or one of the noncanonical poly(A) polymerases from the mouse hippocampus results in a deficit in long term potentiation (LTP) and increase in long-term depression (LTD) (Zearfoss et al., 2008; Udagawa et al., 2012; Mansur et al., 2021).

Neurite-localized transcripts have longer half-lives than somata-enriched isoforms, with average half-lives of mRNAs recorded as 4.8 h and 3.7 h, in neurites and soma cytoplasm of primary cortical neurons, respectively (Tushev et al., 2018; Loedige et al., 2023). The stability and degradation of mRNAs in neurites are crucial for various neuronal functions, including neurite outgrowth and synaptic plasticity. Neurites are an integral part of neuronal communication, and the regulation of mRNA stability in these structures plays a key role in shaping neuronal responses (Figure 2). Several factors contribute to the regulation of mRNA stability and degradation in neurites. Below, we will review the evidence that supports a complex network of RNA-protein interactions underpinning the dynamics of mRNA stability and degradation in neurites.

How do longer 3′ UTRs link with increased stability of local mRNAs? It was postulated that alternative 3′ UTRs have novel and repeated regulatory motifs that might help establish localisation to distal regions of the dendrite or axon (Tushev et al., 2018). RBPs are increasingly found to be essential for transcript stability. Such RBPs, including FMRP, STAUFEN2 (STAU2), and TAR DNA-binding protein 43 (TDP-43), are often found to be associated with their mRNA targets in distal dendritic and axonal branches and synapses (Ortiz et al., 2017; Sharangdhar et al., 2017; Chu et al., 2019). Examples also include many RNA splicing regulators that localize in a bimodal fashion to both the nucleus and neurites, where they facilitate RNA metabolism. Such regulators include the Muscleblind proteins, which regulate alternative splicing in the nucleus (Pascual et al., 2006; Konieczny et al., 2014) and the correct localisation of mRNAs in neurons (Wang et al., 2012; Hildebrandt et al., 2023). Evidence from the nematode Caenorhabditis elegans (C. elegans) indicates that Muscleblind-1 (MBL-1) binds to mRNA transcripts encoding microtubule proteins to regulate their stability. Indeed, microtubule stability in sensory neuron axons is compromised in mbl-1 mutants due to reduced levels of α-tubulin and β-tubulin (Puri et al., 2023). Another well-known splicing regulator also involved in RNA stability is SNRNP70, a core spliceosome protein. SNRNP70 was found to localize to cytoplasmic RNA granules and associate with mRNA transcripts, controlling their axonal trafficking and stability in zebrafish motor neurons, ultimately regulating neuromuscular connectivity (Nikolaou et al., 2022).

The longer half-lives of localized transcripts can also be explained by a lack of destabilization elements. Evidence suggests that neurite-localized mRNAs are depleted of destabilizing elements (Loedige et al., 2023). Such sequences include AU-rich elements (AREs), and those that promote m⁶A (N⁶-methyladenosine) modifications which induce mRNA degradation. It was shown that high mRNA stability is both necessary and sufficient for localisation to neurites, with depletion of mRNA-stabilizing proteins ELAVL and LARP1 interfering with transcript localisation to neurites. Also, alleviation of m⁶A-dependent mRNA degradation by depletion of YTHDF, or removal of destabilizing AREs, were sufficient to increase the stability of transcripts and shift these toward neurites (Loedige et al., 2023).

The most extensively studied mechanism for RNA degradation is by nonsense-mediated RNA decay (NMD), a cellular surveillance mechanism that recognizes and degrades mRNAs containing premature termination codons (PTCs) or nonsense mutations. NMD is a crucial quality control mechanism in eukaryotic cells, ensuring the removal of faulty transcripts and maintaining the integrity of the cellular proteome. The NMD pathway involves a series of proteins and complexes that recognize PTCs and facilitate mRNA degradation. Key components include UPF1, UPF2, and UPF3, which form the core NMD complex. These proteins interact with the exon junction complex (EJC) and other factors to initiate mRNA degradation (Lykke-Andersen and Jensen, 2015). Although NMD is initiated as soon as a PTC is detected in the nucleus, evidence suggests that the pathway can also operate locally to regulate neurite outgrowth, axon guidance and synaptic plasticity through the degradation of selected mRNA isoforms containing NMD-inducing PTCs (see IR RNAs section).

In the hippocampus, the NMD pathway operates within dendrites to regulate synaptic function and plasticity by increasing Glutamate receptor, GLUR1, surface levels (Notaras et al., 2020). UPF2 was shown to promote local synthesis of GLUR1 in dendrites through local NMD-mediated degradation of Arc and Prkag3 mRNAs, whose proteins negatively influence local translation (Notaras et al., 2020). This observation demonstrates that local translation is regulated by mechanisms that control mRNA degradation in dendrites. In addition to its canonical targets, NMD may also degrade mRNAs that do not carry identifiable NMD-inducing features (He and Jacobson, 2015), however, the mechanisms by which NMD recognizes its atypical targets remain unclear. It is also possible that NMD components could act independently of mRNA degradation to promote local protein synthesis. Indeed, UPF1 was found to regulate synaptic plasticity in hippocampal neurons by facilitating the transport and translation of mRNAs through its association with STAU2 (Graber et al., 2017).

IR transcripts are thought to rarely serve a coding function. Retained introns frequently insert PTCs into transcripts, expected to activate transcript degradation by NMD upon the pioneer round of any translation. A well-known example of local translation of an IR mRNA occurs in the developing spinal cord (Chen et al., 2008; Colak et al., 2013). Here, commissural axons are initially attracted to the ventral midline and, upon crossing, become repulsed. Such axon guidance depends on the interaction between axon membrane receptors (Robo proteins) and proteins of the extracellular matrix (Slit proteins) (Jaworski et al., 2010). Following transcription, Robo3 transcripts are processed into either of two isoforms−fully spliced Robo3.1 (no IR), and intron-26-retaining Robo3.2 containing a PTC (Chen et al., 2008; Colak et al., 2013). Prior to reaching the ventral midline, Robo3.1 mRNAs are translated, preventing activation of ROBO1 and ROBO2 that are present at low levels on axons, while Robo3.2 transcripts are translationally repressed. Once the axon has been exposed to floorplate signals in the spinal cord midline, rapid translation of Robo3.2 mRNA is triggered, producing a peptide with a distinct C-terminus compared to the peptide produced from Robo3.1. ROBO3.2 protein increases the ability of ROBO1 and ROBO2 to bind to Slit proteins, which in turn repels the axon from the midline area, allowing appropriate axon positioning (Chen et al., 2008). The ROBO3.2 C-terminus is composed of intron-encoded amino acid residues up to the PTC. As expected, Robo3.2 translation also activates NMD of the transcript, however, this was shown to be functionally important, limiting production of the protein to the correct quantity (Figure 3). Blocking NMD in commissural neurons caused accumulation of Robo3.2 mRNA and ROBO3.2 protein and disproportionate axon repulsion from the midline, indicating the physiological importance of NMD to ensure functionally relevant amounts of protein are synthesized. Thus, NMD drives tight temporal and spatial control of the expression of the protein (Colak et al., 2013).

Other examples of proteins from IR transcripts have also been described. SMN1 functions in spliceosome assembly, implicating it in the splicing process. A specific isoform, aSMN, produced from an mRNA retaining intron-3, is found in axons and is important for axonogenesis (Setola et al., 2007). The specific function/s of the shortened peptide are unclear. In another study, Nxf1, which encodes the nuclear export factor NXF1, produces a transcript that retains intron-10 and undergoes translation to produce the shortened protein isoform, sNXF1, detected in dendrites of rodent cortex (Li et al., 2016). Intron-10 contains a constitutive transport element, which requires NXF1 for nuclear export of the IR mRNA (Li et al., 2006). The authors report a high level of expression of sNXF1 in endogenous adult rodent brain suggesting either the IR transcript does not undergo NMD, or that it is expressed at very high levels.

The extent to which translation occurs more widely from neurite-localized IR mRNAs is unclear. Analyses of neurite ribosome profiling/footprinting data have not explored the extent to which reads map to introns, likely because proportionally they represent very few, owing to NMD activation. Given that PTCs are introduced at some point within introns, reads would be expected to map specifically to the 5′ of introns. However, retained introns could alternatively produce novel peptides by introducing novel translational start sites. An example of this has not yet been reported in neurites. Translation from sequences outside canonical coding regions such as introns, typically produces unstable proteins with hydrophobic tails, either targeted for degradation by the proteasome, or to the membrane (Kesner et al., 2023). However, more stable proteins may be produced from IR transcripts, where retention status is often conserved (Sorek and Ast, 2003; Galante et al., 2004; Buckley et al., 2014), and introns are more GC rich than non-retained introns (Braunschweig et al., 2014).

Retained introns have also been shown to be important for RNA transport to neurites (Buckley et al., 2011; Ortiz et al., 2017; Figure 3). Many retained introns, including Fmr1 intron-1, were shown to contain ID elements with motifs that were previously shown to regulate BC1 ncRNA localisation to dendrites (Buckley et al., 2011). Fmr1 encodes FMRP, which localizes to the soma and dendrites, and is important for proper synaptic plasticity (Richter and Zhao, 2021). Reporters expressing Fmr1 intron-1 ID elements exhibit dendrite localisation, and compete with endogenous IR transcript populations, resulting in altered distribution of the overall population of FMRP protein (Buckley et al., 2011). Mutations in the ID element dramatically reduced dendritic targeting of the reporters, indicating the importance of the sequence to achieve localisation.

STAU2 binds to retained intron-16 of CaMKIIα, required for dendrite localisation of transcripts in mouse hippocampal neurons (Ortiz et al., 2017). Intron-16 retention is conserved in human (Braunschweig et al., 2014) and rat (Buckley et al., 2011), suggesting it may have a conserved role. The authors investigated the fate of IR transcripts under different conditions. Blocking protein synthesis by cycloheximide treatment does not increase intron-16-retaining transcript expression when not undergoing synaptic stimulation, indicating the transcripts are not NMD targets under these conditions. Stimulation with BDNF or NMDA results in decreases in intron-16-retaining CaMKIIα transcripts, which was prevented by cycloheximide treatment, suggestive of translation-dependent degradation by NMD. However, given that overall transcript levels are unaffected by stimulation, one could also hypothesize that the intron-16-retaining portion are instead locally spliced (see Local splicing section below). CaMKIIα protein levels or isoform differences following stimulation were not investigated.

STAU2 has also been shown to be required for the transport of an IR Calm3 mRNA, in dendrites of mature rat hippocampal neurons (Sharangdhar et al., 2017). In this case, the 5′ and 3′ exons flanking the intron are 3′ UTR, sequence classically associated with mRNA transport. Overall, STAU2 was found to strongly bind retained introns within the 3′ UTRs of 28 mRNAs, suggesting the protein similarly regulates the localisation of other transcripts.

Many questions remain regarding the nature of transport granules containing IR RNAs. One hypothesis is that IR transcripts act as the means of transport of fully spliced counterparts occupying the same granule. Such retained introns could also act as a scaffold/platform, binding relevant RBPs, facilitating time or activity sensitive RNA processing of neighboring spliced transcript counterparts. Alternatively, retained introns could act as a scaffold, binding RBPs to catalyze granule organization. Similar roles have been reported for 3′ UTR sequences (Ma and Mayr, 2018; Mayr, 2019).

The local splicing of IR mRNAs could provide a powerful means for the rapid expansion of the pool of translatable mRNAs when needed, or for local decisions to be made on whether to excise introns alone or with a neighboring exon to generate alternative protein isoforms on demand. Direct mechanistic evidence for endogenous local splicing has yet to be shown, and its possibility remains a controversial hypothesis in the field. Despite studies finding an increasing number of splicing factors localized to neurites, spliceosomes are huge and complex structures, and only a small portion of the snRNA and protein components have been detected at substantial levels (Poulopoulos et al., 2019). Below, we discuss the studies that have shown evidence supporting the possibility of local mRNA splicing (Figure 3).

One early study focused on the 6000-nucleotide long retained intron-16 in Kcnma1 transcripts (Bell et al., 2008). Intron-16-retaining transcripts were estimated to form 10% of the total population of Kcnma1 transcripts in rat hippocampal neuron dendrites. Targeting specifically the IR isoform with siRNAs was able to specifically reduce their pools. Significantly lower levels of KCNMA1, a calcium-activated BK channel protein, and perturbed neuronal firing properties were also observed. The authors hypothesized that intron-16 may be locally spliced in dendrites to increase the pool of translatable mRNAs. In a subsequent study by the same group, intron-17 of Kcnma1 was also shown to be retained (Bell et al., 2010). STREX (stress axis regulated exon) is an alternative exon sitting immediately downstream of intron-17. The intron contains regulatory elements controlling the splicing of STREX in response to activity. Inclusion of the exon alters the activity of the channel the protein sits in. In the study, intron-17-retaining mRNAs were detected in dendrites, with the intron either retained alone or in combination with STREX. Knockdown of intron-17-retaining isoforms downregulates STREX-containing isoforms of KCNMA1, most prominently in dendrites, and also disrupts the burst firing abilities of hippocampal neurons. The authors suggested intron-17-retaining isoforms become spliced within dendrites, facilitating the production of STREX-containing KCNM1A. However, the mechanism by which any splicing event would occur is unclear and was not addressed in either study.

Intriguingly, an earlier study by the group indicated canonical splicing capabilities in dendrites of primary cultured rat hippocampal neurons, a process widely accepted as exclusively nuclear (Glanzer et al., 2005). U1 snRNA and splicing factors required for spliceosome assembly were detected by in situ hybridisation and immunohistochemistry, respectively. Dendrites were isolated from somas and transfected with the pre-mRNA splicing construct, chicken δ-crystallin (cdc) mRNA, consisting of a 257-nucleotide intron flanked by exons-14 and -15, with a FLAG sequence in-frame with exon-15. Spliced transfected mRNA was detected in 50% of experiments, with multiple splice junction variants clustering around the canonical donor and acceptor splice sites suggesting classic pre-mRNA splicing. FLAG epitope was also detected in dendrites, which was not possible without local splicing. Another more recent study suggesting canonical splicing occurring outside of the nucleus in neurons, identified that cytoplasmic pools of spliceosomal protein, SNRNP70, a core U1 snRNP component, rescue defects in alternative splicing events in snrnp70 null zebrafish embryos (Nikolaou et al., 2022). Rescued events were enriched in genes associated with neuronal ontologies such as synaptic vesicle recycling proteins.

Although studies thus far have explored the possibility of canonical local splicing, the mechanism may be non-canonical, such as that described at the endoplasmic reticulum (ER) membrane during the unfolded protein response (UPR) (Back et al., 2006; Uemura et al., 2009). The accumulation of incorrectly folded proteins in cells causes ER stress and subsequent activation of the UPR to resolve the situation. This involves upregulated transcription of XBP1, mRNAs of which localize to the ER surface where a 26-nucleotide intron is excised by IRE1, inositol requiring kinase-1, which has endoribonuclease activity. The exposed mRNA 5′ and 3′ fragments are then ligated. Following non-canonical splicing the transcript undergoes translation producing a transcriptional activator of genes involved in the UPR. ER extends into axons and dendrites and could therefore similarly act as a platform for neurite splicing events (Öztürk et al., 2020).

A role in microRNA (miRNA) regulation has been suggested for retained introns in the cytoplasm of motor neurons (Figure 3). A recent study identified that a specific set of introns become transiently retained in the cytoplasm of neural precursor cells during lineage restriction of human iPSC-derived motor neurons (Petrić Howe et al., 2022). Intriguingly, these introns were enriched for 14 miRNA motifs. The authors showed that the IR transcripts are not targets for downregulation by miRNA binding. Conversely, reduced IR transcript expression led to increased expression of predicted miRNA target genes (a readout of miRNA activity). Such findings were not explained by changes in miRNA levels. The authors suggest the retained introns act as sponges, quenching miRNA binding and action on target mRNAs. Intriguingly, the reported retained introns were also enriched for binding capacity of miRNA regulatory proteins, including DROSHA and PUM2. However, loss of DROSHA did not affect levels of IR transcripts suggesting the protein does not process miRNAs from the introns. Regardless, it remains intriguing to hypothesize in other cases that miRNAs could be synthesized locally from introns. Thus far, processing of miRNAs from introns has only been observed in the nucleus (Westholm and Lai, 2011). While the study did not focus on neurite-localized IR transcripts, many miRNAs are known to localize to axons and dendrites (see ncRNAs section below), suggesting similar regulation could be present in neurites.