RBM17 Human

What is RBM17 and what is its fundamental role in RNA splicing?

RBM17 (also known as SPF45) is a splicing factor that plays a critical role in pre-mRNA processing, particularly in a subset of human short introns. Recent research has revealed that the well-established pre-mRNA splicing mechanism involving U2AF heterodimer cannot function efficiently in certain short introns. Instead, a novel mechanism has been discovered where RBM17 forms a complex with SAP30BP to mediate splicing of short introns with truncated polypyrimidine tracts (PPTs) .

The mechanism involves SAP30BP guiding RBM17 to active early spliceosomes, which is particularly important because RBM17 cannot directly bind to truncated PPTs in vitro. This discovery challenges the conventional understanding that U2AF heterodimer (U2AF2–U2AF1) mediates early splicing reactions in all introns regardless of length .

Methodologically, researchers investigating RBM17's fundamental role should employ both knockdown experiments and rescue studies, combined with comprehensive RNA-seq analysis to identify global changes in splicing patterns.

How does RBM17 expression vary across human tissues and what are the implications?

RBM17 expression patterns across human tissues can be analyzed using resources like the Human Protein Atlas. In brain tissue specifically, RBM17 expression data is organized across 13 main brain structures, representing the maximum values found in constituent brain areas .

To properly investigate tissue-specific expression, researchers should:

• Utilize RNA-seq data from resources like GTEx and FANTOM5 to compare expression levels across tissues

• Employ tissue microarrays with validated antibodies for protein-level confirmation

• Consider single-cell RNA-seq to understand cell type-specific expression within tissues

• Correlate expression patterns with tissue-specific splicing events to establish functional relevance

The tissue distribution of RBM17 may help explain tissue-specific splicing regulation and potentially illuminate why certain diseases associated with RBM17 dysfunction affect specific tissues more than others.

How does RBM17 contribute to leukemic stem cell maintenance in acute myeloid leukemia?

RBM17 has been identified as a critical factor in leukemic stem cell (LSC) maintenance in acute myeloid leukemia (AML). Research demonstrates that RBM17 upregulation preferentially marks and sustains LSCs and directly correlates with shortened patient survival .

Experimental evidence shows that RBM17 knockdown in primary AML cells leads to:

• Increased myeloid differentiation

• Impaired colony formation capacity

• Reduced in vivo engraftment potential

Through integrative multi-omics analyses, researchers have discovered that RBM17 repression causes inclusion of poison exons and production of nonsense-mediated decay (NMD)-sensitive transcripts for pro-leukemic factors and the translation initiation factor EIF4A2 . This mechanistic understanding provides a rationale for targeting RBM17 or its downstream NMD-sensitive splicing substrates as a potential AML treatment strategy.

For researchers investigating this area, recommended methodologies include:

• Patient-derived xenograft models to assess effects on LSC function in vivo

• Colony-forming assays to measure self-renewal capacity

• RNA-seq combined with rMATS analysis to identify relevant splicing changes

• Proteomic analysis to confirm downstream effects on translation and ribosome biogenesis

What is the role of RBM17 in glioma progression and what mechanisms underlie its effects?

RBM17 has been found to be overexpressed in glioma patients, and this overexpression correlates with poor prognosis. Research indicates that downregulation of RBM17 suppresses proliferation and induces apoptosis in glioma cells .

The underlying mechanisms appear to involve RBM17's control over apoptosis and proliferation pathways, potentially through its splicing regulatory function. One pathway potentially involved is the Fas receptor signaling pathway, which regulates apoptosis and immune responses .

For researchers investigating RBM17's role in glioma:

• Patient tissue analysis should compare RBM17 expression levels between different grades of glioma and correlate with survival data

• Functional studies should employ both in vitro and in vivo models with RBM17 knockdown or overexpression

• Mechanistic studies should focus on identifying glioma-specific splicing targets of RBM17 using RNA-seq

• Therapeutic potential could be explored by testing RBM17 inhibition in combination with standard glioma treatments

What are the optimal RNA-seq methodologies for investigating RBM17-dependent splicing events?

Based on published research, the following comprehensive RNA-seq methodology is recommended for investigating RBM17-dependent splicing:

• Sample preparation:

  • Perform siRNA-mediated knockdown using two independent RBM17-targeted siRNAs

  • Include appropriate control siRNAs (e.g., universal negative control siRNA)

  • Extract total RNA using high-quality isolation kits (e.g., NucleoSpin RNA kit)

  • Enrich for poly(A) mRNA using magnetic isolation modules

• Library preparation and sequencing:

  • Prepare RNA libraries using directional RNA library prep kits

  • Sequence using high-throughput platforms (e.g., NovaSeq6000) with 150bp paired-end strategy

  • Aim for sufficient depth to detect alternative splicing events (≥30M reads per sample)

• Bioinformatic analysis:

  • Map sequence reads to human genome reference (e.g., hg19) using HISAT2

  • Assemble mapped reads using Stringtie

  • Analyze alternative splicing events using rMATS

  • Define significant changes with FDR <0.05

  • Calculate PPT score and length using specialized software like SVM-BP finder

This methodology has successfully identified RBM17-dependent splicing events in previous studies and provides a robust framework for further investigations.

How can researchers effectively study the RBM17-SAP30BP complex and its functional significance?

To study the RBM17-SAP30BP complex and its functional significance, researchers should employ an integrated approach:

• Interaction characterization:

  • Co-immunoprecipitation to confirm the interaction in different cell types

  • Proximity ligation assays to visualize the interaction in situ

  • Structural studies (X-ray crystallography or cryo-EM) to determine interaction interfaces

• Functional validation:

  • Compare knockdown effects of RBM17 alone versus SAP30BP alone versus double knockdown

  • RNA-seq analysis to identify splicing events dependent on either or both factors

  • Rescue experiments with wild-type and mutant proteins to identify critical interaction domains

• Mechanistic investigation:

  • RNA-protein interaction studies (CLIP-seq) to map binding sites on target RNAs

  • In vitro splicing assays with purified components to reconstitute the activity

  • Live-cell imaging to track the dynamics of complex formation during splicing

This comprehensive approach would provide insights into how the RBM17-SAP30BP complex functions in splicing short introns with truncated polypyrimidine tracts, a process that cannot be effectively carried out by the canonical U2AF heterodimer .

How does the mechanism of RBM17-mediated splicing differ from canonical U2AF-dependent splicing?

The discovery of RBM17-SAP30BP-dependent splicing represents a paradigm shift in understanding pre-mRNA processing. A comprehensive comparison between these mechanisms reveals:

Researchers investigating these differences should:

• Perform comparative binding studies with model pre-mRNAs

• Use mutational analysis to define minimal sequence requirements

• Employ in vitro splicing assays with purified components to directly compare efficiencies

• Analyze the evolutionary conservation of both pathways across species

What computational approaches can identify and characterize RBM17-dependent alternative splicing events?

To effectively identify and characterize RBM17-dependent alternative splicing events, researchers should implement a multi-step computational workflow:

• Data generation:

  • Perform RNA-seq following RBM17 knockdown/knockout

  • Include both control and experimental conditions with sufficient replicates

  • Consider time-course experiments to capture primary versus secondary effects

• Primary analysis pipeline:

  • Quality control and preprocessing of raw reads

  • Alignment to reference genome using splice-aware aligners (HISAT2)

  • Transcript assembly (Stringtie)

  • Alternative splicing analysis (rMATS)

• Advanced characterization:

  • Motif enrichment analysis around affected splice sites

  • RNA secondary structure prediction in affected regions

  • Classification of events by type (exon skipping, alternative 5'/3' splice sites, etc.)

  • Analysis of polypyrimidine tract features using specialized tools like SVM-BP finder

• Integration with other data types:

  • Correlation with RBM17 binding sites from CLIP-seq data

  • Functional annotation of affected genes and pathways

  • Prediction of NMD sensitivity for alternatively spliced transcripts

  • Translation efficiency analysis for affected transcripts

This comprehensive approach enables not only identification of RBM17-dependent events but also mechanistic insights into the rules governing RBM17's splicing activity.

How does RBM17 regulate nonsense-mediated decay through alternative splicing?

Integrative multi-omics analyses have revealed a critical connection between RBM17 and nonsense-mediated decay (NMD). RBM17 repression leads to inclusion of poison exons and production of NMD-sensitive transcripts for pro-leukemic factors and the translation initiation factor EIF4A2 .

This connection presents several research directions:

• Mechanistic investigation:

  • Identify the sequence features of RBM17-regulated poison exons

  • Determine whether RBM17 directly suppresses poison exon inclusion or acts through intermediaries

  • Assess whether RBM17 affects other aspects of NMD beyond regulating poison exon inclusion

• Functional significance:

  • Compare the transcriptome-wide effects of RBM17 knockdown with and without NMD inhibition

  • Identify the subset of RBM17 splicing targets that are most sensitive to NMD

  • Determine whether RBM17-regulated NMD contributes to cancer progression

• Therapeutic implications:

  • Evaluate whether combined inhibition of RBM17 and NMD would have synergistic effects

  • Identify cancer types most susceptible to disruption of this regulatory axis

  • Develop strategies to selectively target RBM17-regulated NMD-sensitive transcripts

For researchers pursuing this question, a combination of transcriptome profiling, functional validation, and mechanistic dissection would be essential to fully understand how RBM17 leverages NMD to maintain cancer cell survival.

What is the impact of SAP30BP-RBM17 interaction on gene expression programs in different cellular contexts?

The discovery that SAP30BP guides RBM17 to active early spliceosomes represents a novel regulatory mechanism in RNA processing . To fully understand the impact of this interaction on gene expression programs:

• Context-dependent interaction analysis:

  • Compare the SAP30BP-RBM17 interaction strength across different cell types

  • Investigate how the interaction is regulated during cell differentiation or stress

  • Determine whether specific signaling pathways modulate the interaction

• Genome-wide splicing impact:

  • Perform comparative RNA-seq analysis after knockdown of either RBM17, SAP30BP, or both

  • Identify splicing events that specifically depend on the interaction versus independent roles

  • Characterize the features of introns most affected by disruption of the interaction

• Downstream effects on gene expression programs:

  • Analyze how disruption of the interaction affects specific cellular pathways

  • Determine tissue-specific consequences of interaction inhibition

  • Identify whether certain disease states show altered SAP30BP-RBM17 interaction

This research would provide valuable insights into how the SAP30BP-RBM17 complex functions as a regulatory hub in RNA processing and potentially identify new therapeutic opportunities.

What strategies could be employed to target RBM17 for cancer therapy?

Given RBM17's critical role in cancer biology, particularly in maintaining leukemic stem cells and promoting glioma progression , several therapeutic strategies could be considered:

• Direct inhibition approaches:

  • Small molecule inhibitors targeting RBM17's RNA binding domain

  • Peptide-based inhibitors disrupting RBM17-SAP30BP interaction

  • Antisense oligonucleotides to downregulate RBM17 expression

  • PROTAC-based degradation of RBM17 protein

• Indirect targeting strategies:

  • Inhibition of downstream effectors like EIF4A2, which is enriched in LSCs

  • Targeting specific RBM17-regulated splicing events critical for cancer maintenance

  • Combination with NMD inhibitors to enhance the effect of poison exon inclusion

  • Synthetic lethality approaches based on cancer-specific RBM17 dependencies

• Considerations for clinical development:

  • Biomarker development to identify patients most likely to respond

  • Evaluation of potential on-target toxicities in normal tissues

  • Assessment of resistance mechanisms that might emerge

  • Rational combination strategies with standard-of-care treatments

The research provides "a rationale to target RBM17 and/or its downstream NMD-sensitive splicing substrates for AML treatment" , and similar approaches could be explored for other cancers like glioma where RBM17 plays a pro-tumorigenic role .

How can researchers develop biomarkers based on RBM17 activity for cancer prognosis?

To develop clinically relevant biomarkers based on RBM17 activity:

• Expression-based biomarkers:

  • Quantify RBM17 mRNA and protein levels in patient samples

  • Correlate expression with clinical outcomes across cancer types

  • Develop standardized assays for clinical implementation

  • Establish threshold values for stratifying patients

• Splicing signature biomarkers:

  • Identify a panel of RBM17-dependent splicing events detectable in patient samples

  • Develop RT-PCR or NanoString-based assays to quantify these events

  • Validate the signature in retrospective patient cohorts

  • Assess prognostic and predictive value in prospective studies

• Functional biomarkers:

  • Develop assays to measure RBM17 activity in patient-derived cells

  • Correlate functional measurements with treatment response

  • Explore liquid biopsy approaches to monitor RBM17 activity during treatment

  • Integrate with other prognostic factors for comprehensive risk assessment

Research has already demonstrated that RBM17 upregulation correlates with shortened patient survival in AML and poor prognosis in glioma , providing a foundation for biomarker development across multiple cancer types.

What are the priority research questions regarding RBM17's role in normal development and disease states?

Several high-priority research questions emerge from the current understanding of RBM17:

• Developmental biology:

  • How does RBM17 expression and function change during embryonic development?

  • What are the consequences of RBM17 knockout in specific lineages during development?

  • Does RBM17-SAP30BP-mediated splicing play tissue-specific roles during organogenesis?

• Disease mechanisms:

  • Beyond cancer, does RBM17 dysregulation contribute to neurodegenerative or cardiovascular diseases?

  • How does RBM17 respond to cellular stress conditions like hypoxia or DNA damage?

  • Are there genetic variations in RBM17 or its binding sites associated with disease susceptibility?

• Regulatory mechanisms:

  • How is RBM17 expression and activity regulated at transcriptional, post-transcriptional, and post-translational levels?

  • What signaling pathways modulate the RBM17-SAP30BP interaction?

  • Does RBM17 have functions beyond splicing regulation, such as roles in transcription or translation?

• Evolutionary perspectives:

  • How conserved is the RBM17-SAP30BP-dependent splicing mechanism across species?

  • Did this mechanism evolve specifically to handle constraints in short introns with truncated polypyrimidine tracts?

  • Are there species-specific adaptations in RBM17 function related to genome complexity?

Addressing these questions will provide a more comprehensive understanding of RBM17's place in cellular physiology and pathology.

What new technologies and methodologies might advance our understanding of RBM17 biology?

Emerging technologies that could significantly advance RBM17 research include:

• Single-cell technologies:

  • Single-cell RNA-seq to reveal cell-type-specific RBM17-dependent splicing events

  • Single-cell proteomics to correlate RBM17 protein levels with cellular phenotypes

  • Spatial transcriptomics to map RBM17 activity in tissue contexts

• Genome engineering:

  • CRISPR-based screening to identify synthetic lethal interactions with RBM17

  • Base editing to introduce specific mutations in RBM17 or its binding sites

  • CRISPRi/CRISPRa for temporal control of RBM17 expression

• Structural biology:

  • Cryo-EM to visualize the RBM17-SAP30BP complex bound to RNA substrates

  • Hydrogen-deuterium exchange mass spectrometry to map protein-protein interaction surfaces

  • Integrative structural modeling to understand complex assembly dynamics

• High-throughput functional assays:

  • Massively parallel reporter assays to characterize RBM17 binding preferences

  • CRISPR tiling screens to identify functional domains within RBM17

  • Synthetic splicing constructs to systematically test sequence requirements

These technological advances would enable researchers to address current knowledge gaps and develop a more comprehensive understanding of RBM17 biology in health and disease.