Systematic investigation of the functional coupling between transcriptional and post-transcriptional regulatory interactions underlying tissue-specific 3'UTRs variability.

Project Information

NeuroUTR

Grant agreement ID: 657749

DOI

10.3030/657749

Project closed

EC signature date 19 March 2015

Start date 1 June 2015

End date 31 May 2017

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 183 454,80

EU contribution

€ 183 454,80

Coordinated by

THE FRANCIS CRICK INSTITUTE LIMITED
United Kingdom

Periodic Reporting for period 1 - NeuroUTR (Systematic investigation of the functional coupling between transcriptional and post-transcriptional regulatory interactions underlying tissue-specific 3'UTRs variability.)

Reporting period: 2015-06-01 to 2017-05-31

Summary of the context and overall objectives of the project

The appropriate functioning of the different tissues that compose our body is enabled by the precise and coordinated expression of specific gene-sets at defined time-point in specific cell types. Disruption in gene expression regulation can lead to various and complex disorders such as neurodegenerative diseases. Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and incurable motor neuron disease (MND) characterised by disruptions in gene metabolism and protein homeostasis. If a growing number of causative gene mutations are being identified in ALS, the aetiology is still unknown and the early molecular events underlying the disease remain poorly understood. As a dramatic consequence, no effective treatments have been developed for this devastating disease that involves paralysis and death within 3-5 years of diagnosis.

In the last 10 years, major steps have been achieved in the development of technologies that enable the simultaneous analysis of all the genes that are expressed in a specific cell type, also called RNA-seq data, shedding light on various cellular mechanisms and disorders. Here we aimed to develop bioinformatics methods and mathematical models to RNA-seq data from neurons of various sources, including human ALS patients, in order to better understand the molecular mechanisms underlying neuronal development and diseases. In doing this, we have re-annotated the boundaries of genes and quantified their usage from RNA-seq data. Next we have applied and expanded existing methods to two distinct RNA-seq data-sets leading to the following key findings: in collaboration with the laboratory of Antonella Riccio (MRC Laboratory for Molecular Cell Biology at University College London) we have uncovered the mechanisms underlying the remodeling in the axons of the products of a gene, the transcripts. In collaboration with Rickie Patani (Francis Crick Institute) and Jernej Ule (Francis Crick Institute), we have identified the exact cascade of gene regulatory events underlying MN development and demonstrated an acceleration of this process in samples derived from ALS patients.

In doing this project we have not only advanced our current knowledge about RNA regulation but we have enabled a better understanding of the regulatory mechanisms underlying ALS, opening in the long term, the future development of new therapies that ameliorate the regulation of genes in neurodegenerative diseases.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

1. Identification of a novel mechanism of 3’ UTR remodeling in axonal compartment
In cells mRNA transport is mediated by elements located within the terminal part of the gene which is not translated into a protein called 3' untranslated region (3’ UTR). Each transcript may express a variety of 3' UTR isoforms. Short 3' UTRs are quite often associated with increased translation however whether 3' UTR length is dynamically regulated and the potential impact on local protein synthesis remain unknown. In collaboration with the laboratory of Antonella Riccio (MRC Laboratory for Molecular Cell Biology at University College London), we aimed to investigate the 3' UTR dynamics between cell body and axons in developing neurons. All wet experiments were performed by Catia Andreassi. We utilized compartmentalized chambers to culture primary sympathetic neurons, allowing the isolation and sequencing of mRNA from distinct subcellular compartments (axons versus cell body).

Prior to sequencing, we performed two rounds of mRNA linear amplification, a protocol which oversamples 3' end termini, and thus makes the data looks similar to polyA-seq data. We therefore developed a bioinformatic pipeline to comprehensively annotate and quantify alternative 3' UTR isoforms in rat using these data. With this novel 3' UTR annotations we found that transcript families transported to axons had longer 3' UTRs. This analysis also revealed that hundreds of short 3' UTR isoforms were highly expressed in axons, some of which were uniquely detected in axons. Experimental analysis of Inositol Monophosphatase 1 (IMPA1) transcript indicated that its 3' UTR is shortened in axons and this remodelling correlates with enhanced translation. We also show that the cleavage of IMPA1 3' UTR is mediated by a multi protein complex that includes Upf1, HuD and Ago2. Thus, our findings demonstrate that local remodelling of 3' UTRs plays a critical role in controlling translation efficiency in axons.

2. Identification of novel changes in transcript structure during motor neurogenesis and the impact of ALS gene background on this process
Early abnormalities in adult-onset degenerative diseases including ALS remain understudied, largely due the inaccessibility of human neuronal cell types at distinct stages of disease development. The objective of this collaborative research study with Rickie Patani (UCL/Crick) and Jernej Ule (Crick) was twofold: resolve the molecular events underlying distinct stages of MN lineage restriction and systematically examine the influence of ALS-causing VCP mutation on this process. In order to achieve this, we integrated the directed differentiation of patient-specific iPSCs into spinal MNs with RNA sequencing and comprehensive bioinformatic examination. All wet experiments have been performed by Claire Hall and Giulia Tyzack (Rickie Patani's lab).

We identify novel transcript structure changes during MN development. In resolving the precise nature of sequential post-transcriptional programs underlying distinct stages of MN differentiation, we show that the timing of these molecular events is perturbed by the ALS-causing VCP mutation. In analyzing additional ALS-related MNs samples, we further show that ALS gene backgrounds such as SOD1 and FUS affect in a similar manner the transcript structure of key RNA splicing regulators that are targeted by the accelerated splicing program in VCP samples. Notably these include SFPQ a key regulator of axonal mRNA localization and axonal development which dysregulated transcript structure may play a key role in axonal degeneration during ALS development.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

1. Novel mechanism in RNA metabolism
3' UTRs isoforms usage between cell body and axonal compartment has never been systematically investigated at genome-wide scale. In the collaborative project with Antonella Riccio’s lab we demonstrate for the first time evidence for mechanisms underlying axonal 3' UTR shortening during neuronal development, a new regulatory principles that will be crucial for the emerging field of RNA genomics.

2. New insights into ALS development
In the collaborative project with Rickie Patani, we show that different modes of alternative splicing regulate distinct stages of lineage restriction from iPSCs to MNs, and that ALS-causing gene mutations modulate a global gene regulatory mechanism in this context that may have important functional consequences for motor neuron integrity. This research underscores the value of integrating the directed differentiation strategies of patient specific iPSCs with RNA-seq to confidently uncover transcriptional events driving development and the initiation of disease. In doing this project we have not only advanced our current knowledge about RNA regulation but we have enabled a better understanding of the regulatory mechanisms underlying ALS, opening in the long term, the future development of new therapies that ameliorate the regulation of genes in neurodegenerative diseases.

Summary of the major findings.