SRRM1 Antibody

What is SRRM1 and what is its cellular function?

SRRM1, also known as SRM160 and POP101, is a nuclear protein containing one PWI domain that belongs to the splicing factor SR family. It has a calculated molecular weight of 102 kDa (904 amino acids) but is typically observed at 120-160 kDa in experimental settings . SRRM1 promotes constitutive and exonic splicing enhancer (ESE)-dependent splicing activation by bridging together sequence-specific SR family proteins (SFRS4, SFRS5, and TRA2B/SFRS10) and basal snRNP factors (SNRP70 and SNRPA1) of the spliceosome . Additionally, it stimulates mRNA 3'-end cleavage independently of exon junction complex formation and binds both pre-mRNA and spliced mRNA 20-25 nucleotides upstream of exon-exon junctions . SRRM1 has low sequence specificity when binding RNA and DNA, showing similar preference for either double- or single-stranded nucleic acid substrates .

Which applications are SRRM1 antibodies commonly used for?

SRRM1 antibodies are validated for multiple research applications, as summarized in the table below:

When selecting an SRRM1 antibody, researchers should ensure the antibody has been validated for their specific application and biological system of interest .

What is the typical molecular weight of SRRM1 observed in Western blots?

Although SRRM1 has a calculated molecular weight of 102 kDa based on its 904 amino acid sequence, the observed molecular weight in Western blots typically ranges from 120-160 kDa . This discrepancy between calculated and observed molecular weight is common for nuclear proteins, particularly those involved in splicing, and likely results from post-translational modifications such as phosphorylation of the numerous serine residues in the protein. When performing Western blot analysis to detect SRRM1, researchers should anticipate bands in this higher molecular weight range rather than at the calculated 102 kDa position . This is consistent across multiple antibodies from different vendors, suggesting this is an intrinsic property of the SRRM1 protein rather than an antibody-specific artifact.

Which species reactivity is available for SRRM1 antibodies?

SRRM1 antibodies offer diverse species reactivity options to support comparative studies across different model systems:

The broad cross-reactivity of certain SRRM1 antibodies suggests high conservation of the protein across species . When selecting an antibody for experimental use, researchers should verify the specific species reactivity for their target organism, as this can vary between different antibody products and clones .

Where is SRRM1 typically localized in cells?

SRRM1 is primarily localized in the nucleus, specifically in nuclear speckles (NS), which are prominent biomolecular condensates involved in RNA processing . Immunofluorescence analysis using SRRM1 antibodies typically reveals a speckled nuclear pattern characteristic of splicing factors. SRRM1, along with SRRM2 and SON, plays an essential role in nuclear speckle formation, although SRRM2 and SON appear to form the core structural components .

When conducting immunofluorescence experiments with SRRM1 antibodies, researchers should expect to observe distinct nuclear speckle staining rather than diffuse nuclear localization . Notably, in experiments where SRRM2 was truncated, SRRM1 staining in nuclear speckles remained intact, indicating that SRRM1 localization is not dependent on the integrity of SRRM2 .

How can SRRM1 antibodies be used to study RNA splicing mechanisms?

SRRM1 antibodies serve as valuable tools for investigating RNA splicing mechanisms through multiple experimental approaches:

• Chromatin Immunoprecipitation (ChIP): SRRM1 antibodies can be used to immunoprecipitate SRRM1-bound DNA regions, helping to identify genomic loci where splicing regulation occurs co-transcriptionally.

• RNA Immunoprecipitation (RIP): Using SRRM1 antibodies for RIP experiments allows identification of RNA sequences directly bound by SRRM1, providing insights into its sequence preferences and target RNAs.

• Co-immunoprecipitation (CoIP): SRRM1 antibodies can precipitate SRRM1 along with its protein interaction partners, revealing the composition of splicing complexes . The search results indicate that SRRM1 bridges sequence-specific SR proteins with basal snRNP factors.

• Immunofluorescence combined with RNA FISH: This approach enables visualization of SRRM1 localization relative to specific RNA transcripts, providing spatial information about splicing regulation.

For effective splicing mechanism studies, researchers should design experiments that combine these approaches with functional readouts of splicing events, such as RT-PCR analysis of alternatively spliced transcripts following SRRM1 knockdown or overexpression .

What is the relationship between SRRM1 and nuclear speckles?

SRRM1 is a component of nuclear speckles (NS), which are membrane-less organelles that serve as storage and assembly sites for splicing factors. According to research findings, SRRM1 functions alongside SRRM2 and SON in forming these structures .

The relationship between SRRM1 and nuclear speckles includes:

• Structural association: While SRRM2 and SON appear to form the core of nuclear speckles, SRRM1 is also essential for proper nuclear speckle organization. Depletion of SON leads to only partial disassembly of nuclear speckles, while co-depletion of SON and SRRM2 causes near-complete dissolution of these structures .

• Marker functionality: SRRM1 antibodies are used as reliable markers for visualizing nuclear speckles in immunofluorescence experiments, alongside other markers like SON, SRRM2, and RBM25 .

• Independent localization: Interestingly, when SRRM2 is truncated (SRRM2 tr10 cells), SRRM1 staining in nuclear speckles remains unaltered, indicating that SRRM1 localization doesn't depend on SRRM2 integrity .

When studying nuclear speckles, SRRM1 antibodies provide a valuable tool for monitoring these structures, particularly in experiments where the commonly used SC35 antibody (which primarily recognizes SRRM2, not SRSF2 as previously thought) might not be suitable due to experimental manipulations of SRRM2 .

How can SRRM1 be used as a biomarker in cancer research?

Recent research, particularly from studies on prostate cancer, reveals that SRRM1 has significant potential as a biomarker in cancer research:

• Circulating biomarker: SRRM1 can be detected in plasma samples, making it a potential non-invasive diagnostic and prognostic biomarker. Studies showed that plasma SRRM1 levels were elevated in prostate cancer patients compared to control individuals .

• Prognostic indicator: High plasma SRRM1 levels were associated with shorter castration-resistant prostate cancer (CRPC)-free survival. Patients with higher SRRM1 levels progressed earlier to CRPC, suggesting SRRM1 could serve as a predictive biomarker for disease progression .

• Molecular correlation: SRRM1 plasma levels positively correlated with androgen receptor (AR) expression levels and AR activity in prostate cancer tissues. Additionally, tissue SRRM1 mRNA levels correlated with AR-V7 expression and activity, as well as with genes associated with resistance to hormonal blockade .

For researchers studying SRRM1 as a cancer biomarker, methodological approaches include:

• ELISA or other immunoassays for measuring SRRM1 levels in patient plasma

• Tissue microarray analysis with SRRM1 antibodies to assess expression in tumor samples

• Correlation analyses between SRRM1 expression and clinical parameters or molecular features

• Kaplan-Meier survival analyses stratified by SRRM1 expression levels

The research indicates that SRRM1 may have broader applications as a biomarker beyond prostate cancer, particularly in cancers where splicing dysregulation plays a pathogenic role .

What experimental approaches can be used to validate SRRM1 antibody specificity?

Validating antibody specificity is crucial for ensuring reliable experimental results. For SRRM1 antibodies, several validation approaches should be employed:

• Western blotting with positive and negative controls:

  • Positive controls: Cell lines known to express SRRM1 (e.g., HeLa, Jurkat, HEK-293)

  • Negative controls: SRRM1 knockdown or knockout cell lines

• Immunoprecipitation followed by mass spectrometry:

  • Perform IP with the SRRM1 antibody

  • Analyze precipitated proteins by mass spectrometry

  • Confirm SRRM1 as the predominant target

• Peptide competition assays:

  • Pre-incubate the antibody with excess immunizing peptide

  • Perform parallel experiments with blocked and unblocked antibody

  • Loss of signal in the blocked condition confirms specificity

• Cross-validation with multiple antibodies:

  • Test multiple antibodies targeting different epitopes of SRRM1

  • Compare staining patterns and band patterns

  • Consistent results across antibodies support specificity

• Genetic validation:

  • Create truncated versions of SRRM1 (similar to the approach used with SRRM2)

  • Test antibody recognition across truncations to map epitope recognition

  • Use CRISPR/Cas9 to delete the epitope region and confirm loss of signal

The importance of thorough validation is underscored by the cautionary example of the SC35 antibody, which was long thought to recognize SRSF2 but actually primarily recognizes SRRM2 .

How does SRRM1 relate to androgen receptor signaling in prostate cancer?

SRRM1 has significant relationships with androgen receptor (AR) signaling in prostate cancer:

• Correlation with AR expression and activity:

  • SRRM1 plasma levels positively correlated with matched tissue AR mRNA levels and AR activity (defined by a set of AR-regulated genes)

  • Tissue SRRM1 mRNA levels positively correlated with AR activity in both TCGA and SU2C cohorts

• Association with AR splicing variants:

  • SRRM1 mRNA levels positively correlated with expression of AR-FL (canonical AR full-length variant)

  • SRRM1 showed stronger correlation with AR-V7 (a splicing variant implicated in castration resistance)

  • SRRM1 levels correlated with the ratio of AR-V7/AR-FL and with AR-V7 activity

• Functional impact on AR signaling:

  • In vivo silencing of SRRM1 in prostate cancer xenografts resulted in reduced AR and AR-V7 expression

  • SRRM1 silencing altered the activity of both AR and AR-V7 by reducing expression of AR- and AR-V7-regulated genes

• Clinical implications:

  • SRRM1 expression was elevated in castration-resistant prostate cancer (CRPC) compared to hormone-sensitive prostate cancer (HSPC)

  • SRRM1 expression correlated with a set of 49 genes associated with resistance to hormonal blockade (ADT resistance score)

These findings suggest that SRRM1 may influence AR signaling by regulating AR splicing, potentially promoting the generation of AR-V7, which is known to contribute to resistance to AR-targeting therapies .

Why might there be discrepancies between calculated and observed molecular weight of SRRM1?

SRRM1 has a calculated molecular weight of 102 kDa based on its 904 amino acid sequence, but is typically observed at 120-160 kDa in Western blot experiments . Several factors can explain this discrepancy:

• Post-translational modifications:

  • SRRM1 belongs to the SR protein family, characterized by serine/arginine-rich domains

  • These serine residues are often extensively phosphorylated, adding significant mass

  • Other modifications like methylation, SUMOylation, or ubiquitination may also occur

• Intrinsically disordered regions (IDRs):

  • Many splicing factors, including SRRM family proteins, contain IDRs

  • These regions often migrate aberrantly on SDS-PAGE due to unusual charge distribution

  • The search results mention IDRs in the related protein SRRM2, and SRRM1 likely has similar structural features

• Technical considerations for optimal resolution:

  • Gel percentage affects migration patterns; lower percentage gels (6-8%) provide better resolution for high molecular weight proteins

  • Running gels at lower voltage for longer times can improve size separation

  • Gradient gels can help resolve proteins with unusual migration patterns

When interpreting Western blot data for SRRM1, researchers should expect bands in the 120-160 kDa range rather than at the calculated 102 kDa size . To confirm band identity, additional controls such as SRRM1 knockdown/knockout samples or detection with multiple antibodies targeting different epitopes are recommended.

How can cross-reactivity with other splicing factors be avoided when using SRRM1 antibodies?

Avoiding cross-reactivity with other splicing factors when using SRRM1 antibodies requires careful experimental design and appropriate controls:

• Antibody selection considerations:

  • Choose antibodies raised against unique regions of SRRM1 that have minimal sequence homology with other splicing factors, particularly other SR proteins

  • Review epitope information: the search results mention antibodies targeting specific amino acid regions (e.g., AA 1-160, AA 21-120, AA 771-820, C-terminus)

  • Select affinity-purified antibodies, which typically have higher specificity than crude antisera

• Validation experiments:

  • Perform Western blots comparing wild-type cells to SRRM1 knockdown/knockout cells

  • Test the antibody on a panel of recombinant splicing factors to assess cross-reactivity

  • Conduct peptide competition assays using specific SRRM1 peptides

• Experimental controls:

  • Include parallel staining with antibodies against other splicing factors to distinguish staining patterns

  • Use cell lines with genetically tagged SRRM1 (e.g., GFP-SRRM1) to confirm colocalization with the antibody signal

  • Consider dual-labeling approaches to distinguish between closely related proteins

• Data interpretation safeguards:

  • Be cautious of signals that persist in SRRM1 knockout conditions

  • Compare results across multiple antibodies targeting different SRRM1 epitopes

  • Verify findings with non-antibody-based approaches (e.g., RNA-seq after SRRM1 knockdown)

The example of the SC35 antibody, which was long thought to recognize SRSF2 but actually primarily recognizes SRRM2, underscores the importance of thorough antibody validation to avoid similar misinterpretations with SRRM1 antibodies .

What controls should be included when using SRRM1 antibodies for immunofluorescence studies?

When conducting immunofluorescence studies with SRRM1 antibodies, the following controls should be included:

• Primary antibody specificity controls:

  • Negative control: SRRM1 knockdown or knockout cells to demonstrate signal specificity

  • Peptide competition control: Antibody pre-incubated with immunizing peptide should show reduced or absent signal

  • Isotype control: Non-specific IgG of the same isotype, concentration, and host species as the SRRM1 antibody

• Secondary antibody controls:

  • Secondary antibody only (no primary antibody) to assess non-specific binding

  • Secondary antibody with unrelated primary antibody to check for cross-reactivity

• Positive controls:

  • Cell lines known to express SRRM1 (e.g., HeLa, as mentioned in the search results)

  • Co-staining with established nuclear speckle markers (e.g., SON)

  • If available, cells expressing tagged SRRM1 (e.g., GFP-SRRM1) to confirm colocalization

• Technical controls:

  • Autofluorescence control: Unstained sample to assess inherent cellular fluorescence

  • Fixation control: Different fixation methods may affect epitope accessibility

  • Permeabilization control: Optimize permeabilization for nuclear proteins

A powerful specificity control approach demonstrated in the research is to use mixed cell populations (e.g., cells with and without SRRM1) imaged side-by-side, allowing direct comparison of staining between positive and negative cells under identical experimental conditions .

How should SRRM1 knockdown/knockout validation experiments be designed?

Designing effective SRRM1 knockdown/knockout validation experiments requires careful consideration of several factors:

• Knockdown approaches:

  • siRNA: Successful in vivo silencing of SRRM1 with siRNA has been demonstrated in xenograft models

  • shRNA: For stable knockdown in long-term experiments

  • ASOs (antisense oligonucleotides): Alternative approach for targeting nuclear RNAs

• Knockout strategies:

  • CRISPR/Cas9: Target early exons to maximize disruption

  • Consider conditional knockout systems if SRRM1 is essential for cell viability

  • Multiple guide RNAs to increase efficiency and reduce off-target effects

• Controls and validation:

  • Non-targeting siRNA/shRNA or non-targeting gRNA controls

  • Multiple independent siRNAs or gRNAs targeting different regions of SRRM1

  • Rescue experiments with siRNA-resistant SRRM1 cDNA to confirm phenotype specificity

• Validation methods:

  • Western blot: Confirm protein depletion using validated SRRM1 antibodies

  • qRT-PCR: Measure mRNA reduction

  • Immunofluorescence: Assess loss of nuclear speckle staining

• Functional readouts to assess:

  • RNA-seq to assess global splicing changes

  • RT-PCR for specific alternative splicing events

  • In cancer models, monitor effects on:

    • AR and AR-V7 expression in prostate cancer models

    • Expression of AR-regulated genes

    • Cell proliferation and tumor growth

A successful approach demonstrated in the research used siRNA-mediated SRRM1 silencing in 22Rv1-derived xenograft tumors, which showed reduced tumor growth within 2 weeks of administration, along with reduced AR and AR-V7 expression and activity .

What are common technical challenges when using SRRM1 antibodies for co-immunoprecipitation?

Co-immunoprecipitation (Co-IP) with SRRM1 antibodies presents several technical challenges that researchers should anticipate and address:

• Nuclear protein extraction:

  • SRRM1 is a nuclear protein associated with chromatin and nuclear matrix

  • Standard lysis buffers may not efficiently extract nuclear proteins

  • Use specialized nuclear extraction protocols with higher salt concentrations or sonication

  • Consider nuclear fractionation before IP to enrich for nuclear proteins

• Preserving protein interactions:

  • Many interactions in splicing complexes are RNA-dependent

  • Decision point: Include RNase treatment to identify direct protein-protein interactions, or omit RNase to preserve RNA-mediated complexes

  • Crosslinking (formaldehyde or UV) can help preserve transient interactions

• Antibody selection:

  • Choose antibodies validated for IP applications (the search results mention specific antibodies validated for IP)

  • Consider the epitope location - antibodies targeting functionally important domains may disrupt protein interactions

  • Amount of antibody: The recommended amount is 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

• Background and specificity:

  • Nuclear extracts often have high background

  • Pre-clear lysates with protein A/G beads before adding antibody

  • Include IgG control IP to identify non-specific binding

  • Consider tandem purification approaches for higher specificity

• Detecting high molecular weight proteins:

  • SRRM1 runs at 120-160 kDa, which can be challenging to transfer efficiently

  • Use gradient gels and semi-dry or wet transfer systems optimized for large proteins

  • Lower percentage acrylamide gels (6-8%) provide better resolution for high MW proteins

Successful IP of SRRM1 has been reported from HeLa cells, suggesting this cell line as a good positive control system for optimizing Co-IP protocols .

What recent studies have identified new roles for SRRM1 in disease pathology?

Recent studies have identified several new roles for SRRM1 in disease pathology, particularly in cancer:

• Prostate cancer biomarker and therapeutic target:

  • 2024 research demonstrated that plasma SRRM1 levels are elevated in prostate cancer patients

  • SRRM1 plasma levels correlated with progression to castration-resistant prostate cancer (CRPC)

  • High SRRM1 expression was associated with shorter castration-resistant PCa-free survival

  • SRRM1 expression correlated with androgen receptor (AR) and AR-V7 activity, suggesting a role in therapy resistance

• Regulation of AR splicing:

  • SRRM1 was found to impact the AR splicing process, potentially affecting the AR-V7/AR-FL mRNA ratio

  • In vivo SRRM1 silencing diminished the growth of prostate cancer tumors and downregulated the expression of AR, AR-V7, and their target genes

  • This suggests a role for SRRM1 in promoting AR-persistent CRPC, distinct from the neuroendocrine differentiation promoted by other SRRM family members (SRRM3, SRRM4)

• Essential nuclear function:

  • Research indicated that SRRM1, along with SRRM2 and SON, plays a role in nuclear speckle formation

  • These structures are important for RNA processing, suggesting that dysregulation of SRRM1 could have widespread effects on gene expression

• Role in RNA splicing network integrity:

  • SRRM1's interaction with other splicing factors suggests its importance in maintaining the integrity of the RNA processing network

  • Disruption of this network could contribute to disease states through aberrant splicing patterns

These findings suggest that SRRM1 may play more diverse roles in disease pathology than previously recognized, particularly in cancer progression and therapy resistance .

How is SRRM1 being investigated as a therapeutic target?

SRRM1 is emerging as a promising therapeutic target, particularly in prostate cancer. Current investigational approaches include:

• In vivo silencing approaches:

  • Research demonstrated that SRRM1 silencing with siRNA in prostate cancer xenograft models reduced tumor growth within 2 weeks of administration

  • A single injection of SRRM1 siRNA resulted in significant tumor growth reduction, suggesting potent anti-tumor effects

• Mechanism of action studies:

  • SRRM1 silencing was shown to reduce the expression of AR and AR-V7, key drivers of prostate cancer progression

  • This led to decreased activity of both AR and AR-V7 by reducing the expression of their target genes

  • Studies noted that SRRM1 silencing "re-sensitizes CRPC cells to enzalutamide in vitro," suggesting potential for combination therapy approaches

• Clinical relevance assessment:

  • The finding that SRRM1 expression is elevated in CRPC vs. HSPC samples supports its relevance in advanced disease

  • Correlation between SRRM1 levels and resistance to hormonal blockade strengthens its potential as a therapeutic target

  • The association with AR-V7, a known mediator of resistance to AR-targeting therapies, suggests SRRM1 inhibition could overcome treatment resistance

• Delivery considerations:

  • The successful in vivo silencing of SRRM1 in xenograft models demonstrates feasibility of targeting this splicing factor

  • Research mentions this was accomplished with "a more clinically relevant in vivo model," suggesting progress toward translational applications

Researchers conclude that SRRM1 "could represent a promising... exploitable therapeutic target for PCa" and suggest that their data "provide valuable new avenues to develop novel strategies to tackle this terrible tumor pathology" .

What is the relationship between SRRM1 and other nuclear speckle proteins like SRRM2 and SON?

The relationship between SRRM1 and other nuclear speckle proteins reveals a complex structural and functional network:

• Structural relationships in nuclear speckles:

  • SON and SRRM2 appear to form the core of nuclear speckles (NS)

  • SRRM1 is also a component of NS but plays a secondary role compared to SON and SRRM2

  • Research shows that "depletion of SON leads only to a partial disassembly of NS, while co-depletion of SON and SRRM2 or depletion of SON in a cell-line where intrinsically disordered regions (IDRs) of SRRM2 are genetically deleted, leads to a near-complete dissolution of NS"

• Co-localization patterns:

  • SRRM1 shows extensive co-localization with other NS markers

  • In experiments where SRRM2 was truncated (SRRM2 tr10 cells), SRRM1 staining in nuclear speckles remained intact, indicating that SRRM1 localization is not dependent on SRRM2 integrity

• Molecular functions:

  • All three proteins (SRRM1, SRRM2, and SON) are involved in RNA processing

  • SRRM1 and SRRM2 both contain RS-dipeptide repeats, which are characteristic of splicing factors

  • SRRM1 promotes constitutive and exonic splicing enhancer (ESE)-dependent splicing activation

• Evolutionary relationships:

  • SRRM1 belongs to the same gene family as SRRM2, SRRM3, and SRRM4

  • Different SRRM family members appear to have distinct roles: SRRM3 and SRRM4 promote neuroendocrine differentiation, while SRRM1 may promote AR-persistent phenotypes in prostate cancer

These relationships suggest that while SRRM1, SRRM2, and SON all contribute to nuclear speckle biology, they have distinct roles and influence different downstream pathways .

How are liquid biopsies being used to measure circulating SRRM1 levels in cancer patients?

Liquid biopsies are being employed to measure circulating SRRM1 levels in cancer patients, with significant potential for clinical applications:

• Detection methodology:

  • Research demonstrates that SRRM1 is detectable in human plasma samples

  • While specific assay details aren't fully described in the available literature, immunoassay approaches such as ELISA are likely used to quantify SRRM1 in plasma

  • Studies have measured SRRM1 levels in plasma from control individuals (n=40) and prostate cancer patients (n=166)

• Clinical applications:

  • Diagnostic potential: Plasma SRRM1 levels were elevated in PCa patients and discriminated between control individuals and PCa patients

  • Prognostic value: High plasma SRRM1 levels were associated with shorter castration-resistant PCa-free survival

  • Predictive biomarker: SRRM1 plasma levels could potentially predict response to hormonal blockade therapy

• Correlation with tissue findings:

  • SRRM1 plasma levels positively correlated with matched tissue AR mRNA levels and AR activity

  • This suggests plasma SRRM1 may reflect underlying tumor biology, potentially serving as a surrogate for tumor tissue analysis

• Advantages highlighted:

  • Non-invasive approach: Researchers note that SRRM1 holds potential as a "non-invasive diagnostic and prognostic biomarker"

  • Cost-effectiveness: Studies mention SRRM1 as a "cost-affordable alternative" compared to other approaches like cell-free DNA, circulating tumor cells, or microRNA determination

Research concludes that plasma SRRM1 could address limitations of PSA testing, which can be inadequate for predicting clinical response to hormonal blockade, providing a rationale for further exploration of SRRM1 as a clinically relevant biomarker in humans .

What advanced imaging techniques are being used to study SRRM1 dynamics in living cells?

While current literature doesn't extensively describe advanced imaging techniques specifically for SRRM1 dynamics in living cells, several approaches are likely applicable and being developed based on related studies:

• Fluorescent protein tagging approaches:

  • Creating TagGFP2-tagged versions of SRRM1 using CRISPR/Cas9-based genome editing (similar to approaches described for SRRM2)

  • Endogenously tagged versions would enable live-cell imaging with minimal disruption to native function

  • Immunofluorescence analysis shows SRRM1 in nuclear speckles, making these structures suitable for dynamic imaging

• Fluorescence Recovery After Photobleaching (FRAP):

  • One reference in the search results mentions "in vitro FRAP reveals the ATP-dependent nuclear mobilization of the exon junction complex protein SRm160"