The timing, dosage and location of gene expression are fundamental determinants of brain architectural complexity. In neurons, this is, primarily, achieved by specific sets of trans-acting RNA-binding proteins (RBPs) and their associated factors that bind to specific cis elements throughout the RNA sequence to regulate splicing, polyadenylation, stability, transport and localized translation at both axons and dendrites. Not surprisingly, misregulation of RBP expression or disruption of its function due to mutations or sequestration into nuclear or cytoplasmic inclusions have been linked to the pathogenesis of several neuropsychiatric and neurodegenerative disorders such as fragile-X syndrome, autism spectrum disorders, spinal muscular atrophy, amyotrophic lateral sclerosis and frontotemporal dementia. This review discusses the roles of Pumilio, Staufen, IGF2BP, FMRP, Sam68, CPEB, NOVA, ELAVL, SMN, TDP43, FUS, TAF15, and TIA1/TIAR in RNA metabolism by analyzing their specific molecular and cellular function, the neurological symptoms associated with their perturbation, and their axodendritic transport/localization along with their target mRNAs as part of larger macromolecular complexes termed ribonucleoprotein (RNP) granules.

Alpha-synuclein (SNCA) accumulation plays a central role in the pathogenesis of Parkinson's disease. Determining and interfering with the mechanisms that control SNCA expression is one approach to limiting disease progression. Currently, most of our understanding of SNCA regulation is protein-based. Post-transcriptional mechanisms directly regulating SNCA mRNA expression via its 3′ untranslated region (3′UTR) were investigated here. Mass spectrometry of proteins pulled down from murine brain lysates using a biotinylated SNCA 3′UTR revealed multiple RNA-binding proteins, of which HNRNPD/AUF1 was chosen for further analysis. AUF1 bound both proximal and distal regions of the SNCA 3′UTR, but not the 5′UTR or CDS. In the nucleus, AUF1 attenuated SNCA pre-mRNA maturation and was indispensable for the export of SNCA transcripts. AUF1 destabilized SNCA transcripts in the cytosol, primarily those with shorter 3′UTRs, independently of microRNAs by recruiting the CNOT1-CNOT7 deadenylase complex to trim the polyA tail. Furthermore, AUF1 inhibited SNCA mRNA binding to ribosomes. These data identify AUF1 as a multi-tasking protein regulating maturation, nucleocytoplasmic shuttling, stability and translation of SNCA transcripts.

Antisense oligonucleotides (ASOs) represent a unique category of therapeutics targeting disease-related RNAs. Since this new therapeutic category emerged, the immediate need to analyze ASOs in clinically relevant biological matrices has led to several methodologies, such as ligand binding assays and imaging techniques. To overcome issues in specificity and provide exact quantitative data for ASOs, a new LC-MS/MS method was developed to analyze in brain tissue a novel 4-10-4 gapmer ASO with the potential for treating Parkinson's disease with phosphorothioated backbone and 2'-O-(2-methoxyethyl) modifications. The sample pretreatment protocol to extract the ASO from brain tissue employed solid phase extraction (SPE) and protein digestion. The LC-MS/MS method was fully optimized, validated and applied to quantify the target ASO in brain tissue samples following an in vivo brain distribution study. The method has a Lower Limit Of Quantification of 1 ng/mg and was applied to the study's samples, demonstrating satisfactory sensitivity and providing valuable information about the ASO's distribution in different brain regions over 45 days.

DNA damage-inducible transcript 4 (DDIT4) is a ubiquitous protein whose expression is transiently increased in response to various stressors. Chronic expression has been linked to various pathologies, including neurodegeneration, inflammation, and cancer. DDIT4 is best recognized for repressing mTORC1, an essential protein complex activated by nutrients and hormones. Accordingly, DDIT4 regulates metabolism, oxidative stress, hypoxic survival, and apoptosis. Despite these well-defined biological functions, little is known about its interacting partners and their unique molecular functions. Here, fusing an enhanced ascorbate peroxidase 2 (APEX2) biotin-labeling enzyme to DDIT4 combined with mass spectrometry, the proteins in the immediate vicinity of DDIT4 in either unstressed or acute stress conditions were identified in situ. The context-dependent interacting proteomes were quantitatively but not functionally distinct. DDIT4 had twice the number of interaction partners during acute stress compared to unstressed conditions, and while the two protein lists had minimal overlap in terms of identity, the proteins’ molecular function and classification were essentially identical. Moonlighting keratins and ribosomal proteins dominated the proteomes in both unstressed and stressed conditions, with many of their members having established non-canonical and indispensable roles during stress. Multiple keratins regulate mTORC1 signaling via the recruitment of 14-3-3 proteins, whereas ribosomal proteins control translation, cell cycle progression, DNA repair, and death by sequestering critical proteins. In summary, two potentially distinct mechanisms of DDIT4 molecular function have been identified, paving the way for additional research to confirm and consolidate these findings.

TIA1 is a broadly expressed DNA/RNA binding protein that regulates multiple aspects of RNA metabolism. It is best known for its role in stress granule assembly during the cellular stress response. Three RNA recognition motifs mediate TIA1 functions along with a prion-like domain that supports multivalent protein-protein interactions that are yet poorly characterized. Here, by fusing the enhanced ascorbate peroxidase 2 (APEX2) biotin-labeling enzyme to TIA1 combined with mass spectrometry, the proteins in the immediate vicinity of TIA1 were defined in situ. Eighty-six and 203 protein partners, mostly associated with ribonucleoprotein complexes, were identified in unstressed control and acute stress conditions, respectively. Remarkably, the repertoire of TIA1 protein partners was highly dissimilar between the two cellular states. Under unstressed control conditions, the biological processes associated with the TIA1 interactome were enriched for cytosolic ontologies related to mRNA metabolism, such as translation initiation, nucleocytoplasmic transport, and RNA catabolism, while the protein identities were primarily represented by RNA binding proteins, ribosomal subunits, and eicosanoid regulators. Under acute stress, TIA1-labeled partners displayed a broader subcellular distribution that included the chromosomes and mitochondria. The enriched biological processes included splicing, translation, and protein synthesis regulation, while the molecular function of the proteins was enriched for RNA binding activity, ribosomal subunits, DNA double-strand break repair, and amide metabolism. Altogether, these data highlight the TIA1 spatial environment with its different partners in diverse cellular states and pave the way to dissect TIA1 role in these processes.

Early separation of atypical Parkinsonian syndromes (APS) from idiopathic Parkinson's disease (iPD) remains challenging. Given that many brain‐biased circular RNAs (circRNAs) are stable and expressed in blood cells, peripheral blood mononuclear cell (PBMC) circRNAs were profiled in 140 individuals (iPD 43, MSA 35, PSP 19, healthy controls 43) using RT‐qPCR across 46 a priori candidates. Group differences were analyzed using the Kruskal–Wallis test with FDR correction; clinical associations were examined using Bonferroni‐corrected Spearman correlations with a bootstrap stability assessment; and discrimination was assessed using logistic regression with nested repeated cross‐validation, calibration analysis, and ROC analysis. Six circRNAs differed across groups after FDR adjustment (SLC8A1_circ_0000994, MAPK9_circ_0001566, MGA_circ_0000591, AGTPBP1_circ_0007162, SFMBT2_circ_0000211, HIPK3_circ_0000284), with median levels lowest in iPD and highest in PSP. Internally cross‐validated multi‐marker panels achieved AUCs of 0.88 for PSP versus controls, 0.78 for PSP versus iPD, and 0.67 for MSA versus iPD. Several circRNAs, including non‐differential ones, showed bootstrap‐stable associations with cognitive, mood, working‐memory, and praxis measures. ENCORI‐ and CircInteractome‐based analyses placed the differentially expressed circRNAs within a shared RBP interaction landscape and identified modest but significant miRNA‐target overlap. Findings indicate that PBMC circRNA patterns aid APS‐iPD differentiation and align with clinical impairment within this cohort; external validation, longitudinal assessment, and mechanistic studies are needed to establish diagnostic performance and biological relevance. image