SF3B14 Human

What is SF3B14 and what are its known alternative names?

SF3B14 is a human protein that functions as a component of the 17S U2 SnRNP complex within the spliceosome. It is also known by several alternative names including SF3B6, SAP14, p14, SF3B14A, CGI-110, HSPC175, and HT006. The protein is relatively small, comprising 125 amino acids and with a molecular weight of approximately 14 kDa . It is a highly conserved protein with homologs found across eukaryotic species, indicating its evolutionary importance in cellular processes . The protein is expressed in various human tissues, with particularly notable expression patterns during embryonic development, especially in neural crest progenitors and developing limbs, which correlates with its role in developmental disorders .

What are the structural domains of SF3B14 and their functions?

SF3B14 contains two RNA-recognition motifs (RRMs) that are critical for its function. The RRM1 domain is required for interaction with SF3B145 (another component of the SF3B complex) and for U2 snRNA binding . Specifically, the α-helical surface of RRM1 interacts with SF3B145 (called Cus1 in yeast), leaving the canonical RNA-binding surface available to bind snRNA. This binding to the 5' end region of U2 snRNA is enhanced when complexed with SF3B145 .

The function of the RRM2 domain is less well-defined, though it has been proposed to mediate pre-mRNA binding . Recent studies suggest that RRM2 may also play a role in transcriptional regulation by interacting with RNA polymerase II . Mutational studies have demonstrated that both RRM domains are essential for viability, as substituting two amino acids within either RRM1 (Y52D/F54D) or RRM2 (C150D/Y152D) resulted in lethality in yeast models .

What is the canonical role of SF3B14 in pre-mRNA splicing?

SF3B14 plays a crucial role in the splicing process as part of the SF3B subcomplex within the 17S U2 SnRNP complex of the spliceosome. The spliceosome is responsible for removing introns from transcribed pre-mRNAs . SF3B14 specifically:

• Participates in early spliceosome assembly

• Mediates recognition of the intron branch site during pre-mRNA splicing

• Promotes the selection of the pre-mRNA branch-site adenosine, which acts as the nucleophile for the first step of splicing

• Directly contacts the pre-mRNA branch site adenosine for the first catalytic step of splicing

• Stabilizes the intron branch site-U2 snRNA duplex, thereby promoting binding of introns with poor sequence complementarity

This sequence-independent binding upstream of the branch site is essential for anchoring U2 snRNP to the pre-mRNA . SF3B14 is also a component of the minor spliceosome involved in the splicing of U12-type introns in pre-mRNAs .

What roles does SF3B14 play beyond pre-mRNA splicing?

Recent evidence indicates that SF3B14 is a versatile protein with multiple cellular functions beyond its canonical role in pre-mRNA splicing. These non-canonical functions include:

• Transcriptional regulation: SF3B14 has been shown to interact directly with RNA polymerase II via its RRM2 domain and regulate Pol II-mediated transcription of specific target genes . This role appears to be independent of its function in the spliceosome.

• Translational control: SF3B14 interacts with p180 at the endoplasmic reticulum (ER) and mediates translational control of secretory proteins . As a cofactor of p180, SF3B14 enhances the association of mRNAs with the ER membrane and the assembly of polyribosomes for certain mRNAs, leading to high-rate biosynthesis of secreted proteins .

• Cell signaling: SF3B14 interacts with the BMPR-IA receptor kinase at the cell membrane and facilitates its internalization . Both the RRM2 domain and C-terminal motif of SF3B14 are required for this interaction, suggesting a role in BMP signaling pathways.

• Centrosome regulation: Recent research indicates that SF3B14 is involved in centrosome regulation, suggesting a role in cell division and chromosome segregation .

These diverse functions highlight the multifunctional nature of SF3B14 and explain its essential role in cellular homeostasis and development.

How does SF3B14 contribute to transcriptional regulation?

SF3B14's role in transcriptional regulation represents a significant non-canonical function that appears to be independent of its role in pre-mRNA splicing. Studies in diverse organisms have provided evidence for this function:

In Arabidopsis, the SF3B14 homolog JANUS directly interacts with RNA polymerase II via its RRM2 domain and regulates Pol II-mediated transcription of specific target genes . Similarly, mouse SF3B14 has been shown to interact with Pol II subunits through its RRM2 domain .

In Xenopus embryos, depletion of SF3B14 disrupted the formation of neural crest progenitors, but surprisingly, pre-mRNA processing of a subset of transcriptional regulators critical for neural crest formation was not altered . Instead, the expression of these genes was significantly reduced or undetectable in SF3B14-depleted embryos, suggesting that SF3B14 regulates their transcription either directly or indirectly .

The mechanism by which SF3B14 recruits Pol II specifically to its target genes remains unclear. It lacks recognizable DNA binding domains, raising the question of whether it functions as part of a mediator complex that bridges transcription regulators with Pol II at specific cis-elements for transcription initiation . This represents an important area for future research.

What protein expression systems are optimal for recombinant SF3B14 production?

For recombinant SF3B14 production, Escherichia coli expression systems have been successfully employed. The recombinant human SF3B14 protein can be produced as a full-length protein (amino acids 1-125) with high purity (>95%) . The protein typically includes a His-tag (MGSSHHHHHH) for purification purposes and is suitable for various analytical techniques including SDS-PAGE and mass spectrometry .

When designing expression constructs for SF3B14, researchers should consider:

• The inclusion of appropriate affinity tags for purification (His-tag being common)

• Expression optimization in E. coli strains optimized for human protein expression

• Careful consideration of buffer conditions for maintaining protein stability

• Whether to include native post-translational modifications, which may require eukaryotic expression systems

For studies requiring domain-specific analyses, expression of individual RRM domains may be advantageous, particularly when investigating specific protein-protein or protein-RNA interactions.

What experimental approaches are effective for studying SF3B14's role in splicing?

To investigate SF3B14's role in splicing, researchers commonly employ the following experimental approaches:

• RNA immunoprecipitation (RIP): To identify RNA species that directly interact with SF3B14, particularly U2 snRNA and pre-mRNA substrates.

• In vitro splicing assays: Using recombinant SF3B14 protein and synthetic pre-mRNA substrates to assess splicing efficiency and fidelity.

• CRISPR-Cas9 gene editing: For generating cellular models with SF3B14 knockdown, knockout, or specific mutations. Complete knockout models may not be viable due to the essential nature of SF3B14 .

• RNA-seq analysis: To identify global changes in splicing patterns following SF3B14 manipulation, particularly focusing on differential exon usage and intron retention.

• Splice-junction microarrays: For targeted analysis of splicing events affected by SF3B14 alterations.

• Structural biology approaches: Including X-ray crystallography and cryo-EM to elucidate the precise positioning of SF3B14 within the spliceosomal complex during various stages of the splicing reaction.

When studying SF3B14's splicing function, it's important to distinguish its direct effects on splicing from its indirect effects via transcriptional regulation, which can complicate interpretation of results.

How can researchers differentiate between SF3B14's splicing and non-splicing functions?

Differentiating between SF3B14's splicing and non-splicing functions presents a significant experimental challenge, as these processes are intimately coupled in time and space. Several approaches can help researchers make this distinction:

• Structure-based mutagenesis: Using the crystal structures of SF3B14 (or its yeast homolog Hsh49), researchers can identify key amino acids required for interaction with the spliceosome . Mutations of these residues can theoretically abolish the interaction with the spliceosome without affecting other functions, such as Pol II recruitment.

• Domain-specific experiments: RRM1 is primarily involved in splicing through interaction with SF3B145 and U2 snRNA, while RRM2 appears to have roles in both splicing and transcription . Domain-specific mutations or truncations can help distinguish these functions.

• Temporal separation: Using inducible systems to manipulate SF3B14 expression or activity, researchers can observe immediate effects (likely direct) versus delayed effects (potentially indirect).

• Cell-type specific analyses: As SF3B14 functions in a cell-type specific manner in translational control , comparing its activity across different cell types can help isolate specific functions.

• Integrated multi-omics approaches: Combining RNA-seq, ChIP-seq (for transcriptional targets), and proteomics can provide a comprehensive view of SF3B14's diverse functions.

• Direct target identification: Techniques like CLIP-seq (cross-linking immunoprecipitation followed by sequencing) can identify the direct RNA targets of SF3B14 in both splicing and transcriptional contexts.

What is the role of SF3B14 in Nager syndrome pathogenesis?

Nager syndrome (NS) is an acrofacial dysostosis characterized by craniofacial and limb malformations. SF3B14 has been identified as a causative factor for NS, with haploinsufficiency of SF3B14 being implicated in the development of this disorder .

The connection between SF3B14 deficiency and NS manifestations appears to involve developmental processes:

• Mouse SF3B14 is highly expressed in limbs and somites during embryonic development (day 11), coinciding with the period of rapid limb development .

• In Xenopus embryos, downregulation of SF3B14 led to a reduced number of neural crest progenitor cells and resulted in hypoplasia of neural crest-derived craniofacial cartilages, partially recapitulating the defects of skeletogenesis seen in NS patients .

• Recent findings suggest that SF3B14's role in translational control, particularly its interaction with p180 at the ER, may be relevant to NS pathogenesis . SF3B14 enhances the association of mRNAs with the ER membrane and the assembly of polyribosomes for certain mRNAs, leading to high-rate biosynthesis of secreted proteins including collagens . Reduced SF3B14 levels in heterozygous SF3B14 individuals might result in impaired collagen secretion, potentially contributing to the skeletal malformations characteristic of NS .

This multi-faceted understanding of SF3B14's role in NS pathogenesis highlights how its diverse cellular functions collectively contribute to complex developmental disorders.

How is SF3B14 involved in cancer progression, particularly hepatocellular carcinoma?

SF3B14 has emerged as a significant factor in cancer progression, with particularly strong evidence for its role in hepatocellular carcinoma (HCC):

• Altered expression: SF3B14 is dramatically up-regulated in HCC compared to non-cancerous tissues . Enhanced SF3B14 expression is positively associated with the occurrence of intrahepatic metastasis and poor prognosis, suggesting that SF3B14 functions as a potential oncogene in HCC .

• Functional impact: Knockdown or depletion of SF3B14 significantly inhibited the proliferation and metastasis of HCC cells both in vitro and in vivo .

• Regulatory mechanisms: The elevated expression of SF3B14 in HCC cells has been attributed to reduced levels of miRNA-133b, which normally inhibits SF3B14 expression . Consistently, the expression of miRNA-133b mimics partially suppressed the ability of SF3B14-overexpressing HCC cells to proliferate and migrate .

• Downstream effects: Overexpression of SF3B14 results in mis-splicing of Kruppel-like factor 4 (KLF4), a tumor suppressor-encoding gene, into a non-functional transcript in cancer cells, thereby promoting tumorigenesis in HCC .

Due to its importance in HCC tumorigenesis, SF3B14 has been suggested as a potential early-stage diagnostic marker for HCC .

How can structural biology approaches advance our understanding of SF3B14 function?

Structural biology approaches hold significant promise for advancing our understanding of SF3B14's diverse functions:

• Structure-function relationships: The crystal structures of yeast Hsh49 (SF3B14 homolog) have already been resolved, revealing key amino acids involved in interactions with other spliceosomal components . Further structural studies of human SF3B14 in various functional contexts could identify critical regions for its different activities.

• Complex formation visualization: Cryo-EM studies of SF3B14 within the spliceosome, transcription complexes, and translational machinery could provide insights into how this single protein participates in multiple cellular processes.

• Domain-specific interactions: Understanding the structural basis for the distinct functions of the RRM1 and RRM2 domains would help elucidate how SF3B14 coordinates its diverse activities.

• Small molecule binding: Structural studies could identify potential binding pockets for small molecules that might selectively modulate specific SF3B14 functions, potentially leading to therapeutic approaches.

• Conformational dynamics: Solution-based structural techniques like NMR could reveal how SF3B14 may undergo conformational changes when transitioning between different functional contexts.

These structural insights would be particularly valuable for distinguishing between SF3B14's splicing and non-splicing functions, which remains a significant challenge in the field .

What are the most promising therapeutic strategies targeting SF3B14 in disease contexts?

Based on SF3B14's involvement in various diseases, several therapeutic strategies could be considered:

• For cancer (particularly HCC):

  • Targeted reduction of SF3B14 expression through antisense oligonucleotides or RNAi approaches

  • Small molecule inhibitors that disrupt SF3B14's interaction with specific oncogenic targets

  • miRNA-133b mimetics to suppress SF3B14 expression, shown to reduce proliferation and migration of HCC cells

• For Nager syndrome:

  • Since NS involves haploinsufficiency of SF3B14, therapies aimed at increasing expression or activity of the remaining functional copy

  • Approaches to compensate for reduced collagen secretion, if this mechanism is confirmed

  • Gene therapy approaches to restore normal SF3B14 levels during critical developmental windows

• For other splicing-related disorders:

  • Splice-switching oligonucleotides that might compensate for SF3B14 dysfunction

  • Small molecules that modulate specific SF3B14 activities without affecting others

The multifunctional nature of SF3B14 presents both challenges and opportunities for therapeutic development. Targeting specific functions while preserving others would be ideal but requires a deeper understanding of the structural determinants of its various activities.

What are the challenges in identifying the complete spectrum of SF3B14 target genes?

Identifying the complete spectrum of SF3B14 target genes presents several methodological and conceptual challenges:

• Functional diversity: SF3B14's involvement in splicing, transcription, and translation means that its target genes may be affected at multiple levels of gene expression regulation . Distinguishing direct from indirect effects requires careful experimental design.

• Cell-type specificity: SF3B14 functions in a cell-type specific manner in translational control , suggesting that its target genes may vary across different cell types and developmental stages.

• Redundancy and compensation: As an essential gene, complete loss of SF3B14 is lethal , necessitating partial depletion approaches that may allow for compensatory mechanisms to mask certain targets.

• Integration of multiple processes: Since transcription and pre-mRNA processing are intimately coupled in time and space , separating SF3B14's effects on these processes for individual genes is technically challenging.

• Target recognition mechanisms: For transcriptional targets, the mechanism by which SF3B14 is recruited to specific genes remains unclear, as it lacks recognizable DNA binding domains . This complicates prediction of potential targets based on sequence features.

Addressing these challenges will require integrated multi-omics approaches, careful analysis of direct protein-RNA and protein-protein interactions, and development of systems that allow for acute and selective perturbation of specific SF3B14 functions.