SRSF1 Human

What is SRSF1 and what are its primary functions in human cells?

SRSF1 is a prototypical SR protein and an oncoprotein that plays pivotal roles in RNA metabolism. It functions primarily as a splicing regulator that activates, modifies, or represses the splicing of mRNA precursors . Beyond splicing, SRSF1 also activates translation in the cytoplasm, creating a bridge between nuclear mRNA processing and cytoplasmic protein synthesis. Research has identified approximately 1500 mRNAs that are translational targets of SRSF1, many encoding proteins involved in cell cycle regulation and chromosome organization . SRSF1 is essential for normal cell function, and its expression is subject to negative autoregulation to maintain homeostatic levels, involving multiple layers of post-transcriptional and translational control .

How is SRSF1 structurally organized?

SRSF1 contains two RNA Recognition Motifs (RRMs) - RRM1 and RRM2 - connected by a flexible inter-RRM linker . This structural arrangement allows for a bimodal mode of interaction with RNA. Solution structure studies of RRM1 bound to RNA reveal that this domain binds preferentially to a CN motif (where N is any nucleotide) . The flexible inter-RRM linker enables RRM1 to bind RNA on both sides of the RRM2 binding site . This structural organization contributes to SRSF1's ability to recognize specific RNA sequences and influence various aspects of RNA metabolism, providing opportunities for structure-based protein engineering approaches.

What is the consensus binding motif for SRSF1?

The consensus binding motif for SRSF1 is a purine-rich sequence. Analysis combining k-mer enrichment with motif discovery in SRSF1 mRNA translational targets has identified a specific motif similar to the one obtained when identifying genome-wide targets of SRSF1 . This motif shows a clear gradient of frequency, being more predominant in CLIP-positive translational targets than in CLIP-negative translational targets . Positional analysis indicates that the motif is preferentially located in the coding DNA sequence (CDS) and to a lesser extent in the 5′UTR of SRSF1 translational targets, compared to mRNAs whose translation is unaffected by increased SRSF1 expression .

How does SRSF1 regulate pre-mRNA splicing?

SRSF1 serves as a key regulator of mRNA splicing, both constitutive and alternative. It functions by:

• Binding to exonic splicing enhancers (ESEs) to promote exon inclusion

• Recruiting components of the core splicing machinery

• Influencing splice site selection in alternative splicing events

In specific contexts, such as Alzheimer's Disease (AD), SRSF1 modifies the formation of Tau cellular mRNA splice variants in hypoxic cells, potentially influencing disease development . Similarly, in CD33 splicing related to late-onset AD risk, SRSF1 acts as a splicing enhancer to promote the inclusion of exon 2, which affects the protein's function in microglia . The SRSF1 binding sequence at the 3' end of exon 2 allows CD33 exon 2 inclusion into the mRNA, suggesting that SRSF1 encourages full-length isoform expression, which has implications for signaling regulation in microglia and AD genetic relationships .

How does SRSF1 couple pre-mRNA splicing and translation?

SRSF1 creates a functional bridge between nuclear pre-mRNA splicing and cytoplasmic translation, demonstrating how these processes can be coordinated. Research has shown that mRNAs displaying alternative splicing changes upon SRSF1 overexpression are also translational targets of SRSF1, strongly suggesting that SRSF1 couples pre-mRNA splicing and translation . The mechanism likely involves:

• SRSF1 binding to specific motifs in pre-mRNA in the nucleus

• Influencing splicing decisions

• Remaining associated with the mature mRNA during export to the cytoplasm

• Enhancing translation of the exported mRNA

This dual functionality allows SRSF1 to exert comprehensive control over gene expression, from processing the primary transcript to determining the efficiency of protein synthesis.

What methods identify SRSF1's translational targets?

To identify SRSF1 translational targets, researchers employ multiple complementary approaches:

• Polysomal Shift Analysis: This technique involves fractionating cell cytoplasm across 10–45% sucrose gradients and isolating RNA from subpolysomal and heavy polysomal fractions from control cells and cells transiently overexpressing SRSF1 . Shifts in mRNA distribution from subpolysomal to polysomal fractions indicate enhanced translation.

• Polysome Index Calculation: This involves calculating the log ratios of polysomal mRNAs versus RNAs in subpolysomal plus polysomal fractions, which measures the proportion/density of each transcript in polysomal fractions .

• Reporter Assays: Luciferase reporters harboring SRSF1 binding sites can measure translational activation induced by SRSF1, demonstrating up to threefold increases in activity with corresponding increases in polysomal/subpolysomal ratios of reporter RNA .

• SILAC Analysis: This mass spectrometry-based technique allows for quantitative comparison of protein abundance between different conditions, confirming that polysomal shifts correlate with increased protein levels .

These approaches have identified approximately 1500 mRNAs as translational targets of SRSF1, with 41% previously identified as bona fide RNA targets by CLIP-seq .

What are the primary translational targets of SRSF1?

High-throughput deep sequencing analysis of polysomal fractions in cells overexpressing SRSF1 has identified approximately 1500 mRNAs as translational targets. These include mRNAs encoding proteins involved in:

• Cell cycle regulation (e.g., CDC27, RBBP8, RBL1)

• Chromosome organization and segregation (e.g., CEP70, NDC80, SMC4, CEP57)

• Cancer-related proteins (e.g., NRAS, R-Ras2)

• Transcription and RNA metabolism

Gene Ontology analysis of direct SRSF1 translational targets reveals enrichment in mRNAs associated with cell cycle, chromosome organization, transcription, and RNA metabolism . Experimental validation through polysome profiling, Western blotting, and SILAC analysis confirms that SRSF1 enhances the translation of these targets, resulting in increased protein levels .

How does SRSF1 regulate cell cycle progression?

SRSF1 plays a crucial role in regulating multiple aspects of the cell cycle, particularly mitotic progression, through translational control of key regulatory proteins. When SRSF1 is depleted, approximately 50% of cells remain arrested in the G2/M phase, demonstrating its importance for normal cell cycle progression . The translational activity of SRSF1 is specifically required for normal mitotic progression, as shown by experiments comparing wild-type SRSF1 with a translationally inactive SRSF1-NRS variant .

Mechanistically, SRSF1 enhances the translation of mRNAs encoding proteins essential for:

• Spindle formation and function

• Kinetochore assembly

• M phase progression

• Chromosome segregation

Key targets include CEP70, NDC80, SMC4, CEP57, CEP170, CBX3, and PDS5B, with experimental validation confirming that SRSF1 promotes their translation and increases their protein levels .

What experimental evidence supports SRSF1's role in chromosome segregation?

Multiple lines of evidence support SRSF1's role in chromosome segregation:

• Polysome Profiling: mRNAs encoding proteins involved in chromosome segregation shift to polysomal fractions upon SRSF1 overexpression but not with a translationally inactive SRSF1-NRS variant .

• Western Blotting: SRSF1 overexpression increases levels of chromosome segregation proteins like CEP70, NDC80, and SMC4, while depletion decreases their levels .

• SILAC Analysis: Quantitative proteomics shows increased abundance of proteins related to chromosome segregation (e.g., CEP170, CBX3, PDS5B) upon SRSF1 overexpression .

• Cell Cycle Analysis: SRSF1 depletion causes approximately 50% of cells to arrest in G2/M phase, suggesting defects in mitotic progression .

• MitoCheck Consortium Findings: Previous research has implicated SRSF1 in mitotic progression .

These findings collectively demonstrate that SRSF1 ensures accurate chromosome segregation through translational control of essential mitotic factors.

How can the structure of SRSF1-RNA complexes be determined?

Determining the structure of SRSF1-RNA complexes involves several complementary techniques:

• Nuclear Magnetic Resonance (NMR) Spectroscopy: This technique has successfully determined the solution structure of SRSF1 RRM1 bound to RNA, revealing preferential binding to a CN motif (where N is any nucleotide) . NMR provides detailed information about interactions between specific amino acid residues and RNA nucleotides.

• Structural Analysis of RNA-Binding Domains: Detailed examination of the RRM domains reveals how they recognize specific RNA sequences and how the flexible inter-RRM linker enables a bimodal mode of RNA interaction .

• Mutational Analysis: Structure-guided mutagenesis can validate key residues involved in RNA binding and help understand the structural basis of binding specificity .

These structural approaches provide insights into SRSF1's mode of action and can guide the design of variants with modified binding properties for potential therapeutic applications.

How can SRSF1 be engineered to modify its binding specificity?

Engineering SRSF1 to modify its binding specificity can be achieved through structure-guided mutagenesis based on detailed structural information:

• Single Residue Mutations: Based on the solution structure of RRM1 bound to RNA, a single glutamate to asparagine mutation (E87N) has been shown to grant the protein the ability to bind to uridines instead of its usual cytosine preference .

• Functional Consequences: This engineered variant (E87N) can activate SMN exon 7 inclusion, a strategy relevant for treating spinal muscular atrophy (SMA) . SRSF1 was shown to be an activator of exon 7 inclusion in SMN1 but not SMN2 due to a C to U nucleotide difference at position +6 of the exon .

• Therapeutic Potential: Such structure-based engineering approaches demonstrate how understanding the molecular details of SRSF1-RNA interactions can lead to designed proteins with modified specificities for potential therapeutic applications .

This exemplifies how structural knowledge can be translated into functional modifications with potential clinical relevance.

What is SRSF1's role in neurodegenerative diseases?

SRSF1 has been implicated in several neurodegenerative diseases through its influence on disease-specific splicing events:

In Alzheimer's Disease (AD):

• SRSF1 modifies the formation of Tau cellular mRNA splice variants in hypoxic cells, potentially influencing AD development .

• SRSF1 and PTBP1 act as splicing enhancers to elevate CD33 exon 2 inclusion, which relates to the risk of developing late-onset AD .

• The SRSF1 binding sequence at the 3' end of exon 2 allows CD33 exon 2 inclusion into the mRNA, affecting microglia signaling regulation .

In Amyotrophic Lateral Sclerosis (ALS):

• SRSF1 acts as a toxicity suppressor of cytoplasmic aggregation of TDP-43 in neurons and glial cells .

• Depletion of SRSF1 inhibits cell death in ALS models .

• Disabling SRSF1's interaction with nuclear export factor 1 (NXF1) suppresses export of pathological C9ORF72 transcripts, reducing neurotoxicity .

These findings suggest SRSF1 as a potential therapeutic target, with approaches such as antisense oligonucleotides or small molecules to block specific SRSF1-RNA interactions being considered for therapeutic implementation .

How does SRSF1 contribute to cancer development?

SRSF1 is recognized as an oncoprotein , and its dysregulation contributes to cancer development through multiple mechanisms:

• Translational Regulation of Oncoproteins: SRSF1 enhances the translation of mRNAs encoding cancer-related proteins such as NRAS and R-Ras2 .

• Cell Cycle Dysregulation: SRSF1 translationally regulates mRNAs encoding cell cycle proteins, including CDC27, RBBP8, and RBL1 . Aberrant expression of these proteins can lead to uncontrolled cell proliferation.

• Chromosome Segregation Control: SRSF1 regulates the translation of proteins essential for accurate chromosome segregation . Dysregulation could potentially lead to genomic instability, a hallmark of cancer.

• Alternative Splicing Regulation: SRSF1 can influence the alternative splicing of genes involved in cancer-related processes, potentially generating protein isoforms that promote tumorigenesis.

The complex role of SRSF1 in controlling gene expression at multiple levels—from splicing to translation—provides insights into how this single factor can have profound implications for cancer development when dysregulated .

How can CLIP-seq identify SRSF1 RNA targets genome-wide?

CLIP-seq (Cross-Linking Immunoprecipitation followed by high-throughput sequencing) methodology for identifying SRSF1 RNA targets involves:

• UV Cross-linking: Cells expressing SRSF1 are irradiated with UV light, creating covalent bonds between SRSF1 and directly bound RNA molecules.

• Immunoprecipitation: SRSF1-RNA complexes are isolated using specific antibodies.

• RNA Recovery and Sequencing: RNA fragments bound by SRSF1 are purified and sequenced.

• Data Analysis: Computational analysis identifies binding sites and consensus motifs.

This approach has successfully identified numerous RNA targets of SRSF1. Notably, 41% of mRNAs identified as translational targets of SRSF1 in polysomal shift experiments were previously identified as bona fide RNA targets by CLIP-seq, indicating they are direct targets . CLIP-seq has been instrumental in defining the RNA binding landscape of SRSF1 and understanding its role in post-transcriptional regulation.

What approaches measure SRSF1's effect on specific splicing events?

Several complementary approaches measure SRSF1's effect on specific splicing events:

• RT-PCR/RT-qPCR: These techniques use primers designed to detect specific splice variants and can quantify changes in isoform ratios upon SRSF1 manipulation.

• Minigene Assays: These involve cloning genomic fragments containing alternatively spliced regions into expression vectors, transfecting them with or without SRSF1, and analyzing splicing patterns.

• Exon-Specific Analysis: In the context of CD33 and AD, researchers have examined how SRSF1 influences the inclusion of specific exons (e.g., CD33 exon 2) and the functional consequences of these splicing decisions .

• Mutagenesis of Binding Sites: Modifying predicted SRSF1 binding sites and observing effects on splicing helps validate direct regulation and understand the mechanism.

• Complementation Studies: Testing whether wild-type SRSF1 but not binding-deficient mutants can rescue splicing phenotypes confirms direct regulation.

These approaches have been applied to study SRSF1's role in various splicing events, including those relevant to neurodegenerative diseases .