Neurons must first establish polarity in order to fulfill their function. They receive signals from other cells via a tree-like network of dendrites, process the signals in their cell body (the soma), and send out the resulting impulses through the axon and its branches. In order to generate and maintain this polarity, the protein patterns of axons and dendrites (the neurites) must be different from those of the soma. Researchers from the Berlin Institute of Medical Systems Biology (BIMSB) at the Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC) have found that targeted localization of messenger RNAs (mRNAs) within the neurites plays the primary role in generating such assymetric protein patterns. The finding appeared in the journal Nature Communications.
“Protein localization within a cell can be achieved in three ways: by actively transporting proteins, by translating uniformly distributed mRNAs into proteins more efficiently at one site than at others, or by supplying mRNA molecules in a particularly high amount to those sites where their protein products are needed,” explains Marina Chekulaeva, who leads the research at the MDC. To work out their significance, Allesandra Zappulo, David van den Bruck, Camilla Ciolli Mattioli, Vedran Franke and colleagues combined a sophisticated neuron separation method with genome-wide analyses.
Local proteome, transcriptome, and translatome
The researchers first differentiated mouse embryonic stem cells into neurons using the transcription factor ASCL1. They grew these neurons on a porous membrane in such a way that the soma stayed on the upper side of the membrane, while the projections branched out on the lower side. This made it possible to isolate the soma from the neurites and investigate each cellular compartment separately.
Next, the researchers subjected the proteome of each compartment to mass spectrometry, resulting in the identification of a total of 7,323 proteins. Some 661 of these proteins were enriched by at least twofold in the neurites as compared to the soma. But how many of them are encoded by neurite-enriched mRNAs? When the research team performed a sequencing analysis of the RNAs and compared the two datasets, they found a clear correlation: 303 out of the 661 proteins enriched in neurites – in other words, about 46 percent – originated from targeted mRNA localization.
To corroborate this finding, the researchers examined the association of ribosomes with mRNA, both those in the soma and those in the neurites, thus capturing snapshots of the translation process. A comparison of these snapshots with those from the proteome and the transcriptome confirmed that the neurite-localized mRNAs are translated into proteins more frequently in neurites than in the soma. The researchers validated this finding by labeling the newly created proteins with non-natural amino acids and visualizing protein synthesis with imaging techniques.
The players of RNA transport
“It was a collaborative effort of several labs to obtain the first genome-wide snapshot of the local transcriptome, proteome and translatome that underlie cell polarity,” says Marina Chekulaeva. The generated datasets provide an invaluable resource for future research. This is all the more true because a number of non-coding RNAs and RNA-binding proteins (RBPs) were found enriched in neurites compared with the soma. They are likely involved in regulating the transport, translation or stability of mRNAs in neurites.
The mRNAs are equipped in their non-coding areas with sequences that specify their transport destination – much like ZIP codes – and are recognized and bound by RBPs as well as presumably by non-coding RNAs. Ensuring that these RNA transports succeed is not only important for the healthy development of neurons; all throughout our lives, it influences how the synapses of our neurons respond to signals. That in turn influences such things as how well we learn and remember.
Neurodegeneration as an RNA disorder
“We know that a number of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) are linked with the defects in RNA splicing and transport,” says Marina Chekulaeva. “So we are now investigating whether and how mRNA localization and local translation change during neurodegeneration.”
Alessandra Zappulo1, David van den Bruck1, Camilla Ciolli Mattioli1, Vedran Franke2, Koshi Imami3, Erik McShane3, Mireia Moreno-Estelles4, Lorenzo Calviello5, Andrei Filipchyk6, Esteban Peguero-Sanchez1,7, Thomas Müller8, Andrew Woehler9, Carmen Birchmeier8, Enrique Merino7, Nikolaus Rajewsky6, Uwe Ohler5, Esteban O. Mazzoni4, Matthias Selbach3, Altuna Akalin2 and Marina Chekulaeva1 (2017):Nature Communications 8(583).
1Non-coding RNAs and Mechanisms of Cytoplasmic Gene Regulation, Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine; 2BIMSB Bioinformatics Platform, Max Delbrück Center for Molecular Medicine; 3Proteome Dynamics, Max Delbrück Center for Molecular Medicine; 4Department of Biology, New York University, New York, USA; 5Computational Regulatory Genomics, Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular MedicineL; 6Systems Biology of Gene Regulatory Elements, Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine; 7Departamento de Microbiología Molecular, Instituto de Biotecnología, UNAM, Cuernavaca, Morelos, Mexico; 8Developmental Biology/Signal Transduction, Max Delbrück Center for Molecular Medicine; 9BIMSB Light Microscopy Platform, Max Delbrück Center for Molecular Medicine. Alessandra Zappulo, David van den Bruck, Camilla Ciolli Mattioli and Vedran Franke contributed equally to this work.