Altered mRNA transport and local translation in iNeurons with RNA binding protein knockdown.

Neurons rely on mRNA transport and local translation to facilitate rapid protein synthesis in processes far from the cell body. These processes allow precise spatial and temporal control of translation and are mediated by RNA binding proteins (RBPs), including those known to be associated with neurodegenerative diseases. Here, we use proteomics, transcriptomics, and microscopy to investigate the impact of RBP knockdown on mRNA transport and local translation in iPSC-derived neurons. We find thousands of transcripts enriched in neurites and that many of these transcripts are locally translated, possibly due to the shorter length of transcripts in neurites. Loss of frontotemporal dementia/amyotrophic lateral sclerosis (FTD/ALS)-associated RBPs TDP-43 and hnRNPA1 lead to distinct alterations in the neuritic proteome and transcriptome. TDP-43 knockdown (KD) leads to increased neuritic mRNA and translation. In contrast, hnRNPA1 leads to increased neuritic mRNA, but not translation, and more moderate effects on local mRNA profiles, possibly due to compensation by hnRNPA3. These results highlight the crucial role of FTD/ALS-associated RBPs in mRNA transport and local translation in neurons and the importance of these processes in neuron health and disease.

Functions of the Bloom Syndrome Helicase N-terminal Intrinsically Disordered Region.

Bloom Syndrome helicase (Blm) is a RecQ family helicase involved in DNA repair, cell-cycle progression, and development. Pathogenic variants in human BLM cause the autosomal recessive disorder Bloom Syndrome, characterized by predisposition to numerous types of cancer. Prior studies of Drosophila Blm mutants lacking helicase activity or protein have shown sensitivity to DNA damaging agents, defects in repairing DNA double-strand breaks (DSBs), female sterility, and improper segregation of chromosomes in meiosis. Blm orthologs have a well conserved and highly structured RecQ helicase domain, but more than half of the protein, particularly in the N-terminus, is predicted to be unstructured. Because this region is poorly conserved across multicellular organisms, we compared closely related species to identify regions of conservation, potentially indicating important functions. We deleted two of these Drosophila-conserved regions in D. melanogaster using CRISPR/Cas9 gene editing and assessed the effects on different Blm functions. Each deletion had distinct effects on different Blm activities. Deletion of either conserved region 1 (CR1) or conserved region 2 (CR2) compromised DSB repair through synthesis-dependent strand annealing and resulted in increased mitotic crossovers. In contrast, CR2 is critical for embryonic development but CR1 is not as important. CR1 deletion allows for proficient meiotic chromosome segregation but does lead to defects in meiotic crossover designation and patterning. Finally, deletion of CR2 does not lead to significant meiotic defects, indicating that while each region has overlapping functions, there are discreet roles facilitated by each. These results provide novel insights into functions of the N-terminal disordered region of Blm.

Mis-spliced transcripts generate de novo proteins in TDP-43-related ALS/FTD.

Functional loss of TDP-43, an RNA binding protein genetically and pathologically linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), leads to the inclusion of cryptic exons in hundreds of transcripts during disease. Cryptic exons can promote the degradation of affected transcripts, deleteriously altering cellular function through loss-of-function mechanisms. Here, we show that mRNA transcripts harboring cryptic exons generated de novo proteins in TDP-43-depleted human iPSC-derived neurons in vitro, and de novo peptides were found in cerebrospinal fluid (CSF) samples from patients with ALS or FTD. Using coordinated transcriptomic and proteomic studies of TDP-43-depleted human iPSC-derived neurons, we identified 65 peptides that mapped to 12 cryptic exons. Cryptic exons identified in TDP-43-depleted human iPSC-derived neurons were predictive of cryptic exons expressed in postmortem brain tissue from patients with TDP-43 proteinopathy. These cryptic exons produced transcript variants that generated de novo proteins. We found that the inclusion of cryptic peptide sequences in proteins altered their interactions with other proteins, thereby likely altering their function. Last, we showed that 18 de novo peptides across 13 genes were present in CSF samples from patients with ALS/FTD spectrum disorders. The demonstration of cryptic exon translation suggests new mechanisms for ALS/FTD pathophysiology downstream of TDP-43 dysfunction and may provide a potential strategy to assay TDP-43 function in patient CSF.

Mis-spliced transcripts generate de novo proteins in TDP-43-related ALS/FTD.

Functional loss of TDP-43, an RNA-binding protein genetically and pathologically linked to ALS and FTD, leads to inclusion of cryptic exons in hundreds of transcripts during disease. Cryptic exons can promote degradation of affected transcripts, deleteriously altering cellular function through loss-of-function mechanisms. However, the possibility of de novo protein synthesis from cryptic exon transcripts has not been explored. Here, we show that mRNA transcripts harboring cryptic exons generate de novo proteins both in TDP-43 deficient cellular models and in disease. Using coordinated transcriptomic and proteomic studies of TDP-43 depleted iPSC-derived neurons, we identified numerous peptides that mapped to cryptic exons. Cryptic exons identified in iPSC models were highly predictive of cryptic exons expressed in brains of patients with TDP-43 proteinopathy, including cryptic transcripts that generated de novo proteins. We discovered that inclusion of cryptic peptide sequences in proteins altered their interactions with other proteins, thereby likely altering their function. Finally, we showed that these de novo peptides were present in CSF from patients with ALS. The demonstration of cryptic exon translation suggests new mechanisms for ALS pathophysiology downstream of TDP-43 dysfunction and may provide a strategy for novel biomarker development.

ProtPipe: A Multifunctional Data Analysis Pipeline for Proteomics and Peptidomics.

Mass spectrometry (MS) is a technique widely employed for the identification and characterization of proteins, personalized medicine, systems biology and biomedical applications. By combining MS with different proteomics approaches such as immunopurification MS, immunopeptidomics, and total protein proteomics, researchers can gain insights into protein-protein interactions, immune responses, cellular processes, and disease mechanisms. The application of MS-based proteomics in these areas continues to advance our understanding of protein function, cellular signaling, and complex biological systems. Data analysis for mass spectrometry is a critical process that includes identifying and quantifying proteins and peptides and exploring biological functions for these proteins in downstream analysis. To address the complexities associated with MS data analysis, we developed ProtPipe to streamline and automate the processing and analysis of high-throughput proteomics and peptidomics datasets. The pipeline facilitates data quality control, sample filtering, and normalization, ensuring robust and reliable downstream analysis. ProtPipe provides downstream analysis including identifying differential abundance proteins and peptides, pathway enrichment analysis, protein-protein interaction analysis, and MHC1-peptide binding affinity. ProtPipe generates annotated tables and diagnostic visualizations from statistical postprocessing and computation of fold-changes across pairwise conditions, predefined in an experimental design. ProtPipe is well-documented open-source software and is available at https://github.com/NIH-CARD/ProtPipe , accompanied by a web interface.