Recombinant Mouse Serine/arginine-rich splicing factor 5 (Srsf5)

What is Serine/arginine-rich splicing factor 5 (Srsf5) and what is its primary function?

Srsf5 is a member of the serine/arginine-rich (SR) protein family that plays critical roles in pre-mRNA splicing. These SR proteins are characterized by one or two RNA recognition motifs at the N-terminus and an arginine/serine-rich (RS) domain at the C-terminus. Srsf5 functions as a splicing regulator that binds to specific RNA sequences to promote or repress the use of particular splice sites, thereby controlling alternative splicing patterns. It recognizes specific RNA motifs and recruits components of the splicing machinery to facilitate spliceosome assembly . Unlike simple RNA binding proteins, Srsf5 can influence splice site selection through both direct interactions with pre-mRNA and protein-protein interactions with other splicing factors.

How is Srsf5 structurally and functionally different from other SR proteins?

Srsf5 shares the characteristic domain organization of SR proteins, including RNA recognition motifs and an RS domain, but has distinct RNA binding preferences and protein interaction partners. While SR proteins like SRSF1 (ASF/SF2) and SRSF2 (SC-35) have been more extensively characterized, Srsf5 has specialized functions in tissue-specific alternative splicing, particularly in cardiac development . Unlike some SR proteins that shuttle continuously between the nucleus and cytoplasm, Srsf5 serves as an intermediate shuttler that facilitates mRNA export from the nucleus to the cytoplasm and maintains mRNA stability . The specific RNA sequence motifs recognized by Srsf5 differ from those of other SR proteins, contributing to its unique role in regulating distinct sets of alternative splicing events.

What are the known target genes and splicing events regulated by Srsf5?

Research has identified several critical targets of Srsf5-mediated splicing regulation:

• Myom1 (Myomesin-1): Srsf5 promotes the alternative splicing of Myom1, a protein that crosslinks myosin filaments to the sarcomeric M-line and maintains structural integrity during contraction. Srsf5 is essential for the developmental switch from embryonic to adult isoforms of Myom1 in cardiac tissue .

• DMTF1 (Cyclin D binding myb-like transcription factor 1): Srsf5 regulates alternative splicing of DMTF1 pre-mRNA by binding to a region between DMTF1β and α acceptor splice sites, promoting the splicing of DMTF1β and γ isoforms rather than DMTF1α. The balance between these isoforms is significant as DMTF1α acts as a tumor suppressor, while DMTF1β exhibits oncogenic properties .

• Sarcomeric genes: RNA-Seq analysis of Srsf5-deficient hearts revealed differential splicing events in genes involved in sarcomere organization, muscle contraction, and heart morphogenesis .

What are the most effective methods for expressing and purifying recombinant mouse Srsf5?

For optimal expression and purification of recombinant mouse Srsf5, a multistep approach is recommended:

• Expression system selection: For functional studies requiring post-translational modifications (particularly serine phosphorylation), mammalian expression systems like HEK293T cells are preferred. For structural studies or applications requiring higher protein yields, bacterial expression systems with codon optimization can be employed using E. coli BL21(DE3) cells.

• Fusion tags: N-terminal 6xHis-tag or GST-tag constructs facilitate purification while maintaining protein activity. For mammalian expression, vectors containing CMV promoters with tetracycline-inducible systems allow controlled expression.

• Purification protocol:

  • Affinity chromatography using Ni-NTA columns for His-tagged proteins

  • Size exclusion chromatography to remove aggregates and degradation products

  • Ion exchange chromatography for higher purity

  • Dialysis into storage buffer containing glycerol and reducing agents

• Quality control: Assess protein purity by SDS-PAGE, verify functionality through RNA binding assays, and confirm phosphorylation status through immunoblotting with phospho-specific antibodies .

How can I validate the splicing regulatory activity of recombinant Srsf5 in vitro?

To validate the splicing regulatory activity of recombinant Srsf5 in vitro, implement the following approaches:

• In vitro splicing assays:

  • Prepare pre-mRNA substrates containing known Srsf5-dependent alternative exons (such as Myom1 or DMTF1)

  • Incubate with HeLa nuclear extracts supplemented with purified recombinant Srsf5

  • Analyze splicing products by RT-PCR or Northern blotting

  • Include concentration gradients of Srsf5 to demonstrate dose-dependent effects

• RNA-protein binding assays:

  • Electrophoretic mobility shift assays (EMSA) with labeled RNA oligonucleotides containing Srsf5 binding motifs

  • RNA immunoprecipitation (RIP) to detect direct interactions between Srsf5 and target RNAs

  • RNA pull-down assays using biotinylated RNA probes followed by Western blotting

• Splicing reporter assays:

  • Construct minigene reporters containing Srsf5-regulated exons and flanking intronic sequences

  • Co-transfect with Srsf5 expression vectors in cell lines with low endogenous Srsf5

  • Analyze splicing patterns through RT-PCR and calculate percent spliced in (PSI) values to quantify Srsf5's effect on exon inclusion/exclusion

What are the key considerations when designing CRISPR-Cas9 knockout models for Srsf5?

When designing CRISPR-Cas9 knockout models for Srsf5, consider the following critical factors:

• Guide RNA design:

  • Target early exons (exons 3-6 have been successfully used) to ensure complete functional disruption

  • Verify guide RNA specificity to avoid off-target effects on other SR proteins

  • Design multiple guide RNAs to increase knockout efficiency

• Validation strategy:

  • Confirm genomic deletion through PCR genotyping

  • Verify complete protein loss via Western blotting with specific antibodies

  • Assess functional consequences through analysis of known splicing targets

• Developmental consequences:

  • Be prepared for potential perinatal lethality, as observed in previous Srsf5 knockout models

  • Consider tissue-specific conditional knockout strategies using Cre-loxP systems for studying adult phenotypes

  • Time point selection is crucial - embryonic day 18.5 (E18.5) has been identified as the latest developmental stage before perinatal lethality

• Alternative approaches:

  • Use inducible shRNA knockdown systems for temporal control if complete knockout is lethal

  • Consider hypomorphic alleles through partial deletions or point mutations in functional domains

  • Implement tissue-specific knockouts to bypass early lethality and study tissue-specific functions

How does Srsf5 deficiency affect cardiac development and function?

Srsf5 deficiency severely impacts cardiac development and function as demonstrated by studies using Srsf5 knockout mice. The effects include:

• Structural abnormalities:

  • Noncompaction of the ventricular myocardium, characterized by a thinner compact layer and thicker reticular myocardial trabeculae

  • Irregular heart structure with a protruding right ventricular apex

  • Defects in myocardial densification and wall thinning

• Functional impairments:

  • Reduced left ventricular systolic function

  • Increased left ventricular internal diameter and volume during systole

  • Reduced ejection fraction and fractional shortening

  • Abnormal electrocardiogram patterns with altered QRS complex

  • Prolonged PR and QT intervals, indicating light atrioventricular block

  • Extremely low QRS wave amplitude and heart rate

• Molecular changes:

  • Elevated expression of brain natriuretic peptide (BNP), a marker of myocardial injury

  • Dysregulated alternative splicing of Myom1, preventing the developmental switch from embryonic to adult isoforms

  • Abnormal expression of genes involved in myofibril organization and function, including Myh8, Neb, and Mylpf

The cardiac phenotype of Srsf5-deficient mice demonstrates that Srsf5-regulated alternative splicing is critical for normal heart development, and its absence leads to cardiomyopathy with lethal consequences around birth.

What is the relationship between Srsf5 expression and cancer progression?

The relationship between Srsf5 expression and cancer progression involves several mechanisms:

• Regulation of tumor-related splicing events:

  • Srsf5 regulates the alternative splicing of DMTF1, a transcription factor with dual roles in cancer

  • Srsf5 promotes the splicing of DMTF1β and γ isoforms over DMTF1α

  • DMTF1α acts as a tumor suppressor by promoting p14ARF expression

  • DMTF1β exhibits oncogenic properties by antagonizing DMTF1α

• Expression correlation in breast cancer:

  • Srsf5 expression positively correlates with DMTF1β/α ratio in breast cancer samples

  • Higher DMTF1β/α ratios are associated with worse patient outcomes

  • Srsf5 may therefore contribute to breast cancer progression by altering the balance of tumor suppressive versus oncogenic DMTF1 isoforms

• Mechanism of action:

  • Srsf5 binds to specific RNA elements located between DMTF1β and α acceptor splice sites

  • This binding displaces other splicing factors (such as SF1) from their proximal binding sites

  • Consequently, SF1 binds to distal sites near β and γ acceptor sites, favoring the splicing of these isoforms

These findings suggest that Srsf5 may contribute to cancer progression by modulating the splicing patterns of genes involved in cell proliferation, apoptosis, and tumor suppression.

What developmental processes are disrupted when Srsf5 is deleted?

Srsf5 deletion disrupts critical developmental processes, especially during late embryonic and early postnatal periods:

• Cardiac development:

  • Ventricular compaction is impaired, leading to noncompaction cardiomyopathy

  • Myocardial maturation and trabeculation are disrupted

  • Sarcomere organization and structural integrity are compromised

• Isoform switching during development:

  • Srsf5 is crucial for developmental isoform transitions, such as the switch from embryonic to adult Myom1 isoforms

  • In Srsf5-deficient mice, this switch fails to occur, resulting in retention of embryonic isoforms inappropriate for postnatal cardiac function

• Perinatal survival:

  • Srsf5 knockout mice exhibit perinatal lethality, with most dying within 24 hours of birth

  • Surviving pups at P1 are pale and fail to feed, lacking milk in their stomachs

  • Some mutants may not breathe at birth, suggesting potential neurological or respiratory defects in addition to cardiac dysfunction

• General development:

  • Srsf5-deficient mice show developmental delay and are smaller than wild-type littermates

  • They have smaller spleens, though most other organs appear grossly normal

  • The heart-to-body weight ratio is not significantly different, suggesting proportional growth retardation

These disruptions highlight the essential role of Srsf5 in regulating alternative splicing events critical for proper developmental transitions and organ maturation.

How can RNA-Seq data be optimally analyzed to identify Srsf5-dependent alternative splicing events?

For optimal analysis of RNA-Seq data to identify Srsf5-dependent alternative splicing events, implement the following methodological approach:

• Experimental design considerations:

  • Include biological replicates (minimum n=3) for both control and Srsf5-deficient samples

  • Use paired-end, strand-specific sequencing with sufficient depth (≥50M reads per sample)

  • Consider developmental time points (e.g., E18.5 and P0) to capture dynamic splicing changes

• Bioinformatic analysis pipeline:

  • Quality control: FastQC followed by adaptor trimming and quality filtering

  • Alignment: Use splice-aware aligners (STAR or HISAT2) with mouse genome reference

  • Alternative splicing analysis: Employ specialized tools such as rMATS, MAJIQ, or VAST-TOOLS

  • Differential expression analysis: DESeq2 or edgeR to identify changes in gene expression levels

• Alternative splicing event categorization:

  • Classify events into five main types: skipped exon (SE), retained intron (RI), mutually exclusive exons (MXE), alternative 5' splice site (A5SS), and alternative 3' splice site (A3SS)

  • Quantify each event using percent spliced in (PSI) values

  • Apply statistical thresholds (typically |ΔPSI| ≥ 10% and FDR < 0.05)

• Functional annotation:

  • Perform Gene Ontology (GO) term enrichment analysis to identify biological processes affected

  • Look for enrichment of specific motifs in regions surrounding differentially spliced events

  • Integrate with other datasets (e.g., CLIP-seq) to identify direct vs. indirect Srsf5 targets

This comprehensive approach will enable researchers to identify the full spectrum of Srsf5-dependent splicing events and their functional implications.

What are the most effective strategies for identifying direct RNA targets of Srsf5?

To effectively identify direct RNA targets of Srsf5, combine the following complementary experimental approaches:

• CLIP-seq methods:

  • iCLIP (individual-nucleotide resolution CLIP) provides single-nucleotide resolution of binding sites

  • PAR-CLIP incorporates photoreactive ribonucleoside analogs for enhanced crosslinking efficiency

  • eCLIP offers improved library preparation methods with reduced technical artifacts

  • Analysis should include stringent peak calling with appropriate controls to distinguish specific binding from background

• Motif discovery and validation:

  • Apply de novo motif discovery algorithms (MEME, HOMER) to CLIP-seq peaks

  • Validate motifs through mutagenesis of predicted binding sites in minigene constructs

  • Use in vitro binding assays with purified recombinant Srsf5 and synthetic RNA oligonucleotides

• Integrative analysis:

  • Compare CLIP-seq data with RNA-seq from Srsf5 knockout/knockdown experiments

  • Filter for events that show both direct binding and splicing changes

  • Consider positional effects: Srsf5 binding in exons typically promotes inclusion, while intronic binding can have position-dependent effects

• Competition and cooperation analyses:

  • Investigate interactions with other splicing factors that may compete for binding sites

  • Examine the presence of binding sites for other factors near Srsf5 binding sites

  • Perform knockdown/overexpression of multiple factors to identify cooperative or antagonistic relationships

This multi-faceted approach will distinguish direct Srsf5 targets from indirect effects and provide mechanistic insights into how Srsf5 regulates alternative splicing.

How can Srsf5 phosphorylation states be manipulated to study their impact on splicing regulation?

Srsf5 phosphorylation state manipulation is a sophisticated approach to studying its splicing regulatory functions:

• Phosphorylation site identification and mutagenesis:

  • Use mass spectrometry to map phosphorylation sites within the RS domain

  • Generate phosphomimetic mutants (serine to aspartate or glutamate substitutions)

  • Create phospho-deficient mutants (serine to alanine substitutions)

  • Develop site-specific phospho-antibodies for monitoring phosphorylation status

• Kinase and phosphatase manipulation:

  • Modulate activity of SR protein kinases (SRPKs) using small molecule inhibitors (e.g., SPHINX)

  • Use SRPK1 or SRPK2 knockdown/overexpression to alter Srsf5 phosphorylation levels

  • Apply phosphatase inhibitors (okadaic acid, calyculin A) to prevent dephosphorylation

  • Express protein phosphatase 1 (PP1) to promote dephosphorylation

• Functional assays:

  • Compare nuclear-cytoplasmic distribution of phosphorylation variants using immunofluorescence

  • Assess RNA binding affinities of differently phosphorylated forms using EMSA or RIP

  • Evaluate splicing regulatory activity using minigene assays with known targets

  • Perform rescue experiments in Srsf5-deficient cells with phosphorylation variants

• Temporal phosphorylation dynamics:

  • Implement inducible expression systems for rapid manipulation of phosphorylation state

  • Use phosphatase-coupled mass spectrometry to monitor dynamic changes in phosphorylation

  • Apply kinase inhibitors at different developmental time points to identify stage-specific requirements

These approaches will provide insights into how phosphorylation regulates Srsf5 function and might reveal potential therapeutic strategies targeting this post-translational modification.

What are the most common pitfalls in generating and validating Srsf5 knockout models?

Researchers frequently encounter these challenges when generating and validating Srsf5 knockout models:

• Genetic compensation and adaptation:

  • Problem: Alternative splicing factors may compensate for Srsf5 loss, masking phenotypes

  • Solution: Perform acute depletion (e.g., with degron systems) to minimize compensation, and analyze expression of other SR proteins to detect compensatory upregulation

• Perinatal lethality limiting adult studies:

  • Problem: Complete Srsf5 knockout results in perinatal death, preventing adult phenotype analysis

  • Solution: Implement conditional knockout strategies with tissue-specific or inducible Cre systems, or develop hypomorphic alleles that retain minimal function

• Incomplete validation of knockout:

  • Problem: Truncated proteins or alternative start sites may produce partial Srsf5 activity

  • Solution: Use antibodies targeting different regions of the protein, perform RT-PCR across multiple exons, and conduct functional assays to confirm complete loss of activity

• Distinguishing direct from indirect effects:

  • Problem: Altered splicing may result from secondary effects rather than direct Srsf5 regulation

  • Solution: Integrate RNA-seq data with CLIP-seq or other binding assays to identify direct targets, and perform acute depletion experiments to capture immediate splicing changes

• Heterogeneity in developmental phenotypes:

  • Problem: Variable penetrance of cardiac and other developmental defects

  • Solution: Increase sample size, control for genetic background effects, and carefully stage embryos to account for developmental timing differences

How can I resolve inconsistencies between in vitro splicing assays and in vivo observations of Srsf5 function?

To resolve inconsistencies between in vitro splicing assays and in vivo observations of Srsf5 function:

• Contextual differences analysis:

  • Compare the protein composition of in vitro splicing extracts versus intact cells

  • Supplement in vitro reactions with additional factors that may be limiting

  • Use extracts from the same tissue/cell type as your in vivo model

• Substrate complexity considerations:

  • In vitro assays typically use simplified minigene constructs lacking genomic context

  • Expand minigene constructs to include more extensive intronic and exonic sequences

  • Test multiple substrate variants with different flanking sequences to capture context-dependent effects

• Post-translational modification status:

  • Assess the phosphorylation state of Srsf5 in your in vitro and in vivo systems

  • Pre-treat recombinant Srsf5 with relevant kinases before in vitro assays

  • Apply phosphatase inhibitors if dephosphorylation is suspected in extracts

• Concentration and stoichiometry adjustments:

  • Titrate Srsf5 concentrations in in vitro assays to physiologically relevant levels

  • Consider relative abundances of competing or cooperating splicing factors

  • Measure endogenous Srsf5 levels in target tissues to guide in vitro conditions

• Alternative approaches to bridge the gap:

  • Utilize cell-based splicing assays as an intermediate between in vitro and in vivo systems

  • Perform nuclear extract depletion and reconstitution experiments

  • Develop organoid or ex vivo tissue culture systems that better recapitulate the in vivo environment

This systematic approach will help reconcile discrepancies and develop a more unified understanding of Srsf5 function across experimental systems.

What are the key considerations when interpreting RNA-Seq data from Srsf5-deficient models?

When interpreting RNA-Seq data from Srsf5-deficient models, consider these key factors:

• Primary versus secondary effects:

  • Early time points after Srsf5 depletion more likely reflect direct effects

  • Compare alternative splicing changes with Srsf5 binding data from CLIP-seq

  • Look for enrichment of known Srsf5 binding motifs near differentially spliced regions

• Developmental stage influences:

  • Different developmental stages show distinct patterns of alternative splicing

  • The P0 stage shows more differential splicing events (583) compared to E18.5 (162) in Srsf5 knockout mice

  • Certain splicing events may be stage-specific and depend on developmental context

• Event type distribution analysis:

  • Skipped exon (SE) events typically predominate (364 events at P0)

  • Consider the distribution of other event types: retained intron (RI), mutually exclusive exons (MXE), alternative 5' splice site (A5SS), and alternative 3' splice site (A3SS)

  • Overlap between different event types in the same genes suggests complex regulatory mechanisms

• Impact on gene expression:

  • Splicing changes may lead to nonsense-mediated decay and altered gene expression

  • 661 differentially expressed genes were identified in P0 hearts of Srsf5 knockout mice

  • Enrichment analysis revealed myofibril as a highly affected cellular component

• Tissue-specific effects:

  • Cardiac tissue shows specific sensitivity to Srsf5 deficiency

  • Different tissues may exhibit distinct splicing patterns and gene expression changes

  • Consider tissue-specific expression of other splicing factors that may interact with Srsf5

This comprehensive analytical approach will enable more accurate interpretation of RNA-Seq data from Srsf5-deficient models, leading to more reliable biological insights.

How might Srsf5 be targeted therapeutically in cardiac diseases or cancer?

Potential therapeutic targeting of Srsf5 in cardiac diseases or cancer involves several promising strategies:

• Small molecule modulators:

  • Develop compounds that modulate Srsf5 activity rather than completely inhibiting it

  • Screen for molecules that selectively interrupt Srsf5 interaction with specific RNA targets

  • Target kinases that phosphorylate Srsf5 to modulate its activity indirectly

• RNA-based therapeutics:

  • Design antisense oligonucleotides (ASOs) that block Srsf5 binding to specific targets

  • Develop splice-switching oligonucleotides (SSOs) to redirect splicing of key Srsf5 targets

  • Use siRNA or shRNA approaches for tissue-specific Srsf5 knockdown

• Cardiac disease applications:

  • Target Srsf5 activity during critical developmental windows to prevent noncompaction cardiomyopathy

  • Modulate Srsf5 function to promote proper Myom1 isoform expression in cardiac disorders

  • Develop therapies that compensate for Srsf5 deficiency by targeting downstream effectors

• Cancer therapeutic strategies:

  • Manipulate DMTF1 splicing to favor tumor-suppressive DMTF1α over oncogenic DMTF1β

  • Combine Srsf5 inhibition with conventional chemotherapies in breast cancer models

  • Develop biomarkers based on Srsf5 expression and activity to guide personalized treatment

• Delivery considerations:

  • Employ tissue-specific delivery systems (nanoparticles, viral vectors)

  • Consider temporal aspects of treatment to minimize developmental side effects

  • Develop methods for monitoring splicing changes as pharmacodynamic biomarkers

These approaches offer potential for developing targeted therapies that modulate specific Srsf5-dependent splicing events relevant to disease pathogenesis.

What is the role of Srsf5 in the regulation of circular RNA formation?

The emerging role of Srsf5 in circular RNA (circRNA) regulation presents an exciting frontier:

• Mechanistic contributions:

  • SR proteins including Srsf5 can promote back-splicing events that generate circRNAs

  • Srsf5 binding to exonic splicing enhancers may facilitate the proximity of downstream splice donors to upstream splice acceptors

  • Competition between canonical splicing and back-splicing is likely regulated by Srsf5 concentration and binding affinity

• Experimental approaches:

  • Circular RNA sequencing (circRNA-seq) in Srsf5-deficient versus control samples

  • RNA immunoprecipitation to identify circRNAs directly bound by Srsf5

  • Minigene constructs to test Srsf5's effect on specific back-splicing events

  • CRISPR-mediated manipulation of Srsf5 binding sites in circRNA-producing genes

• Functional implications:

  • CircRNAs can serve as miRNA sponges, potentially affecting post-transcriptional regulation

  • Some circRNAs may be translated into proteins with distinct functions

  • Dysregulation of circRNA formation in Srsf5-deficient hearts may contribute to cardiac phenotypes

• Tissue-specific considerations:

  • CircRNA expression shows high tissue specificity, with brain and heart expressing diverse circRNAs

  • Srsf5's role in circRNA formation may be particularly relevant in these tissues

  • Developmental regulation of circRNAs may intersect with Srsf5's role in developmental transitions

This research direction represents a largely unexplored aspect of Srsf5 function with potential implications for understanding both normal development and disease mechanisms.

How does Srsf5 function integrate with non-coding RNA regulatory networks?

The integration of Srsf5 function with non-coding RNA regulatory networks reveals complex regulatory mechanisms:

• Interactions with microRNAs:

  • Srsf5 may regulate alternative splicing of primary miRNA transcripts, affecting mature miRNA production

  • Srsf5-regulated alternative splicing can create or eliminate miRNA binding sites in 3' UTRs

  • Experimental approaches: small RNA-seq in Srsf5-deficient models, analysis of miRNA binding site gain/loss in alternatively spliced regions

• Long non-coding RNA interactions:

  • Srsf5 may directly bind and regulate lncRNA processing and function

  • Some lncRNAs may act as decoys for Srsf5, modulating its availability for pre-mRNA splicing

  • Certain lncRNAs could function as scaffolds, bringing Srsf5 into proximity with specific pre-mRNAs

• Splicing-related non-coding RNAs:

  • Small nuclear RNAs (snRNAs) forming the core of the spliceosome may interact with Srsf5

  • Srsf5 could modulate efficiency of snRNA-pre-mRNA base-pairing

  • U1 snRNP recruitment to 5' splice sites may be facilitated or inhibited by Srsf5 in a context-dependent manner

• Research methodology:

  • CLIP-seq analysis focused on non-coding RNA interactions

  • RNA-RNA interactome studies to map three-way interactions between Srsf5, target pre-mRNAs, and regulatory ncRNAs

  • Functional studies using antisense oligonucleotides to disrupt specific Srsf5-ncRNA interactions

This integrated view of Srsf5 within broader non-coding RNA networks provides a framework for understanding its extended regulatory functions beyond direct pre-mRNA splicing.

How does the function of mouse Srsf5 compare to its human ortholog?

The comparative analysis of mouse and human Srsf5 reveals important functional conservation and divergence:

• Sequence and structural conservation:

  • Mouse and human Srsf5 share high sequence homology (>95% amino acid identity)

  • Both contain the characteristic RNA recognition motifs and RS domain

  • Phosphorylation sites in the RS domain show strong conservation, suggesting preserved regulatory mechanisms

• Functional similarities:

  • Both mouse and human Srsf5 regulate alternative splicing of target pre-mRNAs

  • They share many target genes, including those involved in cellular processes such as DMTF1 pre-mRNA processing

  • The basic mechanism of splicing regulation through binding to RNA sequence elements is conserved

• Species-specific differences:

  • Certain target exons show species-specific regulation, reflecting evolutionary divergence

  • Human SRSF5 has been more extensively implicated in cancer-related splicing events

  • Mouse Srsf5 knockout studies have predominantly revealed developmental cardiac defects that have not been as well characterized in human systems

• Experimental considerations:

  • Human and mouse cell lines may exhibit different baseline splicing patterns for shared targets

  • When interpreting results from mouse models for human disease relevance, these differences should be considered

  • Validation of key findings in both species is recommended for translational research

This comparative understanding is essential for translating findings from mouse models to human disease contexts and for developing therapeutic strategies targeting Srsf5 or its regulated splicing events.

What are the functional differences between Srsf5 and other SR protein family members?

Functional differences between Srsf5 and other SR protein family members include:

• RNA binding specificity:

  • Srsf5 recognizes distinct RNA sequence motifs compared to other SR proteins

  • While SRSF1 (ASF/SF2) preferentially binds purine-rich sequences, Srsf5 has different sequence preferences

  • These binding differences result in regulation of distinct subsets of alternative splicing events

• Developmental roles:

  • Srsf5 knockout mice exhibit specific cardiac defects and perinatal lethality

  • In contrast, SRSF1 knockout causes embryonic lethality at an earlier stage

  • SRSF2 tissue-specific deletion affects different cell types (particularly hematopoietic cells)

  • Each SR protein appears to have specialized developmental functions despite structural similarities

• Regulatory mechanisms:

  • Srsf5 can both activate and repress splicing depending on binding position relative to regulated exons

  • Srsf5 functions as an intermediate shuttling protein between nucleus and cytoplasm

  • Some SR proteins (like SRSF1) have more pronounced roles in additional processes such as mRNA export, NMD, and translation

  • Phosphorylation dynamics and responsiveness to specific kinases varies among SR family members

• Pathological implications:

  • Srsf5 has specific connections to cardiac development and breast cancer progression

  • Other SR proteins have been implicated in different disease contexts: SRSF2 in myelodysplastic syndromes, SRSF1 in various cancers

  • These differences highlight the non-redundant functions of SR proteins despite their structural similarities

Understanding these functional differences is crucial for targeting specific SR proteins in disease contexts and for interpreting phenotypes resulting from manipulation of individual family members.

How do tissue-specific expression patterns of Srsf5 correlate with its functional significance?

The tissue-specific expression patterns of Srsf5 strongly correlate with its functional significance:

• Cardiac expression and function:

  • Srsf5 is highly expressed in developing cardiac tissue

  • This expression pattern correlates with its essential role in heart development and function

  • Knockout mice show severe cardiac defects including noncompaction cardiomyopathy and systolic dysfunction

  • Srsf5 regulates cardiac-specific splicing events, particularly the developmental switch in Myom1 isoforms

• Comparative tissue expression analysis:

  • Analysis of RNA-seq data across tissues reveals differential Srsf5 expression patterns

  • Tissues with higher Srsf5 expression generally show more Srsf5-dependent alternative splicing events

  • The ratio of Srsf5 to other splicing factors in different tissues may determine splicing outcomes

• Developmental regulation:

  • Srsf5 expression levels change during development in a tissue-specific manner

  • These changes correlate with developmental transitions in alternative splicing patterns

  • For example, the switch from embryonic to adult cardiac isoforms of target genes coincides with changes in Srsf5 activity

• Pathological contexts:

  • Altered Srsf5 expression in breast cancer correlates with changes in DMTF1 splicing

  • The DMTF1β/α ratio positively correlates with Srsf5 expression in breast cancer samples

  • This correlation has functional consequences for cancer progression, highlighting the significance of Srsf5 in this disease context

These correlations between expression patterns and functional significance provide insights into the tissue-specific roles of Srsf5 and help identify contexts where therapeutic targeting might be most effective.