Functions of the SAFB family identified by iCLIP

DNA genes can be translated into one or more messenger RNAs (mRNA) which in turn express a unique protein. The total number of genes encoded in the human genome is similar in number to those found in lower species (e.g. mouse). However, the processes governing gene expression in humans are far more complex than that found in lower species and many more mRNAs (and therefore proteins) are produced from a single gene. This complexity of expression is particularly evident in neuronal cells and the altered functioning of the proteins that govern this process may underlie human neurological diseases. We have found that a protein (SAFB1) not previously known to be expressed in neuronal cells is involved in regulating the processing of specific RNAs. Furthermore, we have identified a novel protein with very similar properties to SAFB1 and called it SAF-like modulator (SLTM). To identify what RNA molecules these proteins interact with we are proposing to use a powerful technique called individual-nucleotide resolution cross-linking and immunoprecipitation (iCLIP). This technique allows: (i) the RNA molecules being bound by SAFB1 and SLTM to be identified in a highly quantitative manner; (ii) the genes and proteins they encode to be identified. Following the identification of these mRNAs we will analyse in detail how SAFB1 and SLTM govern the expression of these mRNA and the proteins they encode. Preliminary studies using this iCLIP technique have shown that SAFB1 governs the levels of expression of specific and important neurological genes. Importantly, it was also shown to influence the levels of an RNA that forms an assembly point in the cell for many of the proteins that govern RNA processing.
This investigation will identify how SAFB1 and SLTM influence the expression of genes that governs important processes in neurons, e.g. memory formation. Many of the neuronal (SAFB regulated) genes already identified are associated with complex human neurological conditions and the results of our study will therefore yield valuable and novel insights into mechanisms that cause human neurological diseases.

Despite the clear evidence for their interaction with a wide range of fundamental cellular processes, the precise function of SAFB proteins has remained difficult to elucidate. We therefore used individual-nucleotide resolution cross-linking and immunoprecipitation (iCLIP) to gain insight into SAFB1 functions and found that it binds: (i) MALAT1 a key regulator of alternative splicing; (ii) specific RNA transcripts, a high proportion of which are transcribed from genes involved in neuronal and synaptic function. Subsequent experiments showed there was an inverse relationship between SAFB1 and MALALT1 levels and confirmed SAFB1 regulated the expression of specific RNA transcripts.
To investigate the function of SAFB1 and SLTM further we will carry out further iCLIP analyses in neuronal cultures derived from human stem cells and in post-mortem tissue. We will use powerful lentiviral mediated knockdown and quantitative measures of RNA (e.g. qPCR) to investigate the function of SAFB1 in relation to effects on splicing and expression of the identified targets. In addition, we will complement these studies using exon arrays to analyse splicing patterns. In the course of these studies we will assess the effect of SAFB1 and/or MALAT1 knockdown on known interacting proteins and nuclear bodies involved in processing of pre-mRNAs. We will also investigate the relationship between MALAT1 and SAFB1 using RNA Fluorescence in situ hybridization (to monitor MALAT1 expression) and immunocytochemical techniques to measure the expression pattern of SAFB1 and associated splicing proteins. We will investigate the physiological relevance of SAFB1's control of RNA transcript expression using techniques already established in our laboratories; including confocal microscopy and lentiviral transduction to measure indices of synaptic function, and neurite outgrowth assays.

We are using cutting edge Individual-nucleotide resolution cross-linking and immunoprecipitation (iCLIP) technology to study the functions of the SAFB family of RNA binding proteins. We have found that SAFB1 is expressed at high levels in neurons and: (i) interacts with an important long non-coding RNA (lncRNA) that regulates pre-mRNA processing; (ii) binds specific RNA transcripts many of which encode proteins involved in neuronal plasticity and function. Furthermore, the role of SAFB proteins in regulating the expression and function of genes involved in synaptic plasticity, receptor expression and neurite outgrowth during development and in mature neurons will be studied. Prior to this work it was not known that SAFB1 was expressed in post-mitotic neurones and regulated their gene expression. Hence, it is highly likely that this work will be of interest to academic and industry based researchers studying neurodegenerative and neuropsychiatric illnesses. We are currently exploring collaboration with a pharmaceutical company to develop a mouse knockout model to explore our findings in relation to human neurological disease.
Hence this research will benefit: (i) local researchers interested in using the powerful iCLIP technology; (ii) the RNA and neuroscience research community; (iii) those in the field of educational science; (iv) the research staff employed on the grant will benefit from training iCLIP technology, and in multidisciplinary approaches to understanding gene function as well as training in transferable skills; (v) members of the general public with an interest in brain function and disease; (vi) Industrial partners. Many of the genes regulated by SAFB1 have been found to be implicated in the development of human neurological illnesses. Identifying novel mechanisms by which they are regulated will therefore be of interest to the pharmaceutical industry allowing potential new targets to be identified and new animal models of disease to be developed.
How will they benefit?
(I, ii & iii) The research will be disseminated through peer reviewed journals within the standard timescale. The research will be presented to the scientific community at national and international conferences. The impact of the research will also be increased once disseminated to our existing collaborators (and upon the establishment of new collaborations): these include collaborations within Bristol (School of Physiology and Pharmacology, School of Clinical Sciences, Frenchay Hospital), Cardiff University Medical School, the Heath Hospital and our extensive network of industrial collaboration and with the MRC Laboratory for Molecular Biology, Cambridge. The applicants have experience in presenting the research to the Media, scientific community and wider public audiences including school children through public engagement seminars organised by Bristol Neuroscience and via local Bristol Cancer charities.
(iv) Staff employed on the project will be trained to use the specific scientific techniques necessary for the successful completion of the project. Staff will also gain a number of transferable skills such as time/project management; communication skills training to scientific and general audiences through public engagement opportunities; team working and networking.
(vi) The findings from this project in the short term will allow us to explore the relationship between the mechanisms governing gene expression and how altered function could lead to the formation of human neurological disease. Such knowledge will be of interest to the public and will be displayed at annual Bristol science fairs (in Broadmead and at @Bristol) and if possible to audiences of school-age children and interested adults.

(v) Before the end of this grant I would envisage our findings will also be of interest to pharmaceutical companies seeking to identify new therapeutic targets and to develop powerful animal models of human neurologic disease.