LSM12 Human

What is LSM12 and what protein family does it belong to?

LSM12 is a member of the Sm-like (Lsm) protein family, specifically one of the larger Lsm proteins that contains an extra C-terminal non-Lsm domain in addition to the canonical Lsm domain. The human LSM12 has a predicted protein size of 21.7 kDa but runs at an observed size of approximately 25 kDa on protein gels . LSM12 belongs to the larger Lsm proteins group (including Lsm11, 12, 14A, 14B, and 16 in humans) that differ from smaller Lsm proteins (Lsm1-10) which contain only an Lsm domain . According to The Human Protein Atlas, LSM12 RNA is ubiquitously and abundantly expressed across different tissues and cell lines, suggesting its fundamental importance in cellular processes .

What is the subcellular localization of LSM12?

Immunofluorescence studies have shown that LSM12 is broadly present in the cytoplasm of wild-type cells. This cytoplasmic localization has been confirmed through immunofluorescence staining with anti-LSM12 antibodies that recognize the C-terminal region of the protein . When LSM12 is knocked out, the staining pattern disappears, confirming the specificity of the localization pattern. Upon transient expression of recombinant LSM12 (with Myc and FLAG tags), the protein replenishes the cytoplasmic expression pattern in LSM12-knockout cells .

How is LSM12 gene expression regulated in different tissues?

Analysis of LSM12 expression using database resources like GEPIA (Gene Expression Profiling Interactive Analysis) has revealed that LSM12 is significantly upregulated in many types of human malignant tumors compared to paired normal tissues . This upregulation pattern has been specifically documented in Oral Squamous Cell Carcinoma (OSCC), where LSM12 shows strong staining throughout tumor tissues (93% of cases) while being negative or weakly expressed in paired normal tissues . The ubiquitous expression of LSM12 RNA across different normal tissues suggests that while baseline expression is maintained in healthy tissues, dysregulation occurs in pathological states, particularly in cancer development.

How does LSM12 function as an NAADP receptor?

LSM12 has been identified as a nicotinic acid adenine dinucleotide phosphate (NAADP) receptor that is essential for NAADP-evoked calcium release. Research using proteomic approaches identified LSM12 as a protein that interacts with both NAADP and two-pore channels (TPCs) . The binding interaction between LSM12 and NAADP occurs at the Lsm domain of the protein, as demonstrated through binding assays with recombinant protein fragments . When LSM12 is knocked out in HEK293 cells, NAADP-evoked calcium release is abolished, confirming its essential role in this signaling pathway. Moreover, microinjection of purified recombinant human LSM12 protein (~4 μM in pipette) together with NAADP immediately restores NAADP-evoked calcium release in LSM12-knockout cells, providing direct evidence for LSM12's function as an NAADP receptor .

What experimental approaches can be used to study LSM12-mediated calcium signaling?

Multiple experimental approaches have been employed to investigate LSM12's role in calcium signaling. These include:

• Knockout/knockdown studies: CRISPR/Cas9 genome editing to generate LSM12-knockout cell lines or siRNA-mediated knockdown to reduce LSM12 expression .

• Calcium imaging: Using calcium-sensitive fluorescent indicators to measure intracellular calcium changes in response to NAADP with or without LSM12 expression .

• Protein-protein interaction studies: Combining pull-down assays with mass spectrometry to identify proteins that interact with LSM12, TPCs, and NAADP. This approach can utilize differential isotope labeling (heavy 13C vs. light 12C) to quantify specific interactions .

• Microinjection assays: Direct introduction of purified proteins and/or signaling molecules into cells to assess functional rescue of calcium signaling .

• Recombinant protein expression: Using bacterial expression systems (e.g., E. coli) to produce purified LSM12 for in vitro binding studies and functional rescue experiments .

These methodologies provide complementary approaches to investigate different aspects of LSM12's function in calcium signaling pathways.

How do two-pore channels (TPCs) interact with LSM12 in NAADP-mediated calcium signaling?

LSM12 has been identified as an interacting protein shared by TPC1, TPC2, and NAADP through proteomic analysis . The interaction between LSM12 and TPCs was confirmed through multiple experimental approaches:

• Pull-down assays with anti-FLAG antibodies from TPC1-FLAG and TPC2-FLAG expressing cells showed the presence of LSM12 in the TPC-interactomes as verified by immunoblotting .

• Similarly, pull-down assays with immobilized NAADP from both TPC1- and TPC2-expressing cells demonstrated the presence of LSM12 in NAADP-interactomes .

• Mass spectrometry analysis with differential isotope labeling (13C vs. 12C) showed high peak ratios (≥3) for LSM12 peptides, indicating its enrichment in test samples compared to negative controls .

These findings suggest that LSM12 functions as an NAADP receptor that mediates NAADP's interaction with TPCs, forming a functional complex that regulates calcium release from lysosomes. This complex formation is essential for NAADP-evoked calcium signaling, as demonstrated by the loss of NAADP-induced calcium release in LSM12-knockout cells .

What evidence suggests LSM12 could be a biomarker for cancer?

Research has identified LSM12 as a potential biomarker for cancer, particularly in Oral Squamous Cell Carcinoma (OSCC). Several lines of evidence support this role:

• High-throughput analysis using mRNA microarrays in a golden hamster carcinogenic model of OSCC identified LSM12 as significantly upregulated during tumorigenesis .

• Validation in clinical samples using immunohistochemistry on tissue microarrays composed of 101 cases of OSCC tissues and paired adjacent normal tissues showed dramatic differences in LSM12 expression. In paired normal tissues, LSM12 staining was negative (91%, 92/101) or weak, while in OSCC tissues, the positive rate was 100%, with strong staining spreading over the whole tissues in 93 cases (93/101, 92%) .

• Functional studies demonstrated that LSM12 overexpression significantly promoted OSCC cell growth, colony formation, migration, and invasion abilities, while LSM12 knockdown inhibited these malignant phenotypes and significantly reduced tumor formation in vivo .

• Analysis using the GEPIA database revealed that LSM12 expression is significantly upregulated in many types of human malignant tumors compared to paired normal tissues, suggesting a broader role in tumorigenesis beyond OSCC .

These findings collectively suggest that LSM12 could serve as a potent biomarker for cancer diagnosis and potentially as a therapeutic target.

How does LSM12 contribute to neurodegeneration pathways?

LSM12 has been implicated in neuroprotective pathways, particularly in the context of C9ORF72-associated amyotrophic lateral sclerosis (ALS). The LSM12-EPAC1 (exchange protein directly activated by cyclic AMP 1) axis defines a neuroprotective pathway that sustains the nucleocytoplasmic RAN gradient . Specifically:

• LSM12 and its downstream effector EPAC1 establish a robust nucleocytoplasmic gradient of RAN-GTP, which is essential for proper cellular function .

• In the context of C9ORF72-derived poly(GR) protein toxicity, which is associated with ALS, LSM12 depletion significantly increased the relative proportion of cells harboring poly(GR)-induced stress granules (SGs) .

• This effect contrasts with LSM12-depletion phenotype in arsenite-induced SGs, indicating that LSM12 has stress-specific effects on cellular stress responses .

• The biochemical association of LSM12 with ATXN2 (Ataxin-2) is conserved between Drosophila and humans, suggesting an evolutionarily conserved role in neuronal function .

These findings suggest that LSM12 plays a complex role in neurodegeneration, potentially through its effects on nucleocytoplasmic transport, stress granule formation, and interaction with known neurodegeneration-associated proteins like ATXN2.

What cellular stress response pathways involve LSM12?

LSM12 has been implicated in multiple cellular stress response pathways, with distinct effects depending on the type of stress:

• In poly(GR)-induced stress (relevant to C9ORF72-associated ALS), LSM12 depletion significantly increases the formation of stress granules (SGs), suggesting that LSM12 normally suppresses this specific stress response .

• Contrastingly, in arsenite-induced stress, LSM12 depletion has a different effect on SG formation, indicating that LSM12's role in stress responses is context-dependent and stress-specific .

• LSM12 functions in the maintenance of nucleocytoplasmic transport through its effects on the RAN-GTP gradient, which is critical for cellular homeostasis under stress conditions .

• In Drosophila, LSM12 recruits the translation factor TWENTY-FOUR to the ATXN2 protein complex and induces translation of the circadian clock gene period, suggesting a role in cellular timing mechanisms that may influence stress response timing .

These diverse roles suggest that LSM12 functions as a stress-response modulator, with its effects varying depending on the specific stress stimulus and cellular context.

How can researchers generate and validate LSM12 knockout or knockdown models?

Several approaches have been used to generate and validate LSM12 knockout or knockdown models:

• CRISPR/Cas9 genome editing: Researchers have generated HEK293 LSM12-knockout cell lines using chemically modified sgRNA (5'-CCAGAAUGUCCCUCUUCCAG-3') and GeneArt Platinium Cas9 nuclease transfected together into cells using Lipofectamine CRISPRMAX Cas9 transfection reagent . The resulting mutations in the Lsm12 alleles included either 1 bp deletion or 68 bps insertion at the start of Lsm12's exon 3, both resulting in framing errors after amino acid residue S46 and truncation of 149 amino acid residues .

• siRNA knockdown: LSM12 expression has been knocked down in SK-BR-3 cells (a breast cancer cell line) using predesigned dicer-substrate siRNA (DsiRNA) targeting exon 3 of LSM12, with a non-targeting DsiRNA used as a negative control .

• Animal models: LSM12 mutant mice have been generated using CRISPR/Cas9 methods via pronuclear injection of a mixture of sgRNA and Cas9 into embryonic stem cells. These models include the Lsm12Δ45-50 mutant mouse line, from which mouse embryonic fibroblasts (MEFs) have been isolated for further study .

Validation of these models typically involves:

• Immunoblotting with anti-LSM12 antibodies to confirm absence or reduction of LSM12 protein expression .

• Immunofluorescence staining to visualize the loss of LSM12 expression in knockout/knockdown cells .

• Functional assays, such as calcium imaging, to confirm the loss of LSM12-dependent functions .

• Rescue experiments with exogenous recombinant LSM12 to confirm that observed phenotypes are specifically due to LSM12 loss .

What protein-protein interaction methods are most effective for studying LSM12 complexes?

Multiple protein-protein interaction methods have been successfully employed to study LSM12 complexes:

• Affinity purification coupled with mass spectrometry (AP-MS): This approach has been used to identify proteins that interact with both TPCs and NAADP. By expressing tagged proteins (e.g., TPC1-eGFP-FLAG or TPC2-eGFP-FLAG) and performing pull-downs with either anti-FLAG antibodies or immobilized NAADP, researchers identified LSM12 as a shared interacting partner .

• Differential isotope labeling for quantitative proteomics: The use of heavy (13C6-Arg/Lys) and light (12C6-Arg/Lys) isotope labeling of proteins allows for quantitative comparison of specific interactions versus background binding. This approach revealed high MS peak ratios (≥3) for LSM12 peptides, confirming its specific enrichment in pull-down samples .

• Immunoprecipitation followed by immunoblotting: This technique has been used to confirm interactions identified by mass spectrometry. For example, immunoblotting confirmed the presence of LSM12 in TPC-interactomes pulled down by anti-FLAG antibody from TPC1-FLAG and TPC2-FLAG expressing cells .

• Recombinant protein binding assays: The use of purified recombinant proteins allows for the testing of direct interactions in vitro, as demonstrated by the use of purified recombinant human LSM12 protein (hLsm12-HisE.coli) expressed in E. coli and purified with immobilized metal affinity chromatography .

These complementary approaches provide robust evidence for protein-protein interactions involving LSM12 and help elucidate the components of functional complexes in which LSM12 participates.

What calcium imaging techniques are optimal for studying LSM12-mediated calcium signaling?

Several calcium imaging techniques have been employed to study LSM12-mediated calcium signaling:

• Fluorescent calcium indicators: While specific details aren't provided in the search results, traditional approaches often use chemical calcium indicators like Fura-2 or Fluo-4 to measure intracellular calcium changes in response to NAADP or other calcium-mobilizing agents .

• Genetically encoded calcium indicators: For analysis in mouse embryonic fibroblasts (MEFs), AAV1 particles carrying CAG-driven GCaMP6f calcium sensor have been used to visualize calcium dynamics. After viral transduction for 16-24 hours, cells were analyzed 24-48 hours after medium change to remove the virus .

• Microinjection-coupled calcium imaging: This approach involves microinjection of signaling molecules (like NAADP) with or without purified proteins (like recombinant LSM12) directly into cells while simultaneously monitoring calcium levels. This technique was successfully used to demonstrate that microinjection of purified hLsm12-HisE.coli protein together with NAADP immediately restored NAADP-evoked calcium release in LSM12-knockout cells .

• Calcium imaging in different cellular models: LSM12's role in calcium signaling has been studied across multiple cell types, including HEK293 cells, SK-BR-3 breast cancer cells, and mouse embryonic fibroblasts, showing that LSM12 is functionally critical for NAADP-evoked calcium release in diverse cell types .

These techniques allow for the real-time visualization and quantification of calcium signals in living cells, providing insights into the spatiotemporal dynamics of LSM12-mediated calcium responses under various experimental conditions.

How does the Lsm domain of LSM12 contribute to its NAADP binding capacity?

The Lsm (Sm-like) domain of LSM12 is a critical structural feature that contributes to its function as an NAADP receptor. While the search results don't provide specific details about the molecular mechanisms of NAADP binding to the Lsm domain, several key insights can be drawn:

• LSM12 belongs to the larger Lsm proteins that contain an Lsm domain plus an extra C-terminal non-Lsm domain . This domain organization suggests that the Lsm domain may provide the primary binding site for NAADP.

• The Lsm protein family is known for RNA binding functions in many contexts, which suggests that the Lsm domain of LSM12 may have evolved to recognize the nucleotide-based structure of NAADP.

• The observed size of LSM12 protein (~25 kDa) is close to the previously reported doublet protein band sizes (22/23 kDa) of putative NAADP receptors photolabeled by [32P]5-azido-NAADP in cell lysates, supporting the identification of LSM12 as an NAADP receptor .

Further research using domain mapping, point mutations within the Lsm domain, and structural biology approaches would be valuable to precisely define how the Lsm domain recognizes and binds NAADP at the molecular level.

What is the relationship between LSM12's roles in RNA processing and calcium signaling?

• As a member of the Lsm protein family, which typically functions in RNA metabolism, LSM12 may have dual functions in both RNA processing and calcium signaling .

• In Drosophila, LSM12 recruits the translation factor TWENTY-FOUR to the ATXN2 protein complex and induces translation of the circadian clock gene period . This suggests that LSM12's role in RNA translation regulation may intersect with its role in signaling pathways.

• LSM12 has been identified as an RNA-splicing factor in the context of cancer research, where it was shown to regulate alternative splicing of certain genes. For example, LSM12 overexpression caused the inclusion of USO1 exon 15, while LSM12 knockdown induced exon 15 skipping .

• The functional implications of LSM12-mediated splicing were demonstrated when the exon 15-retained USO1 significantly promoted malignant phenotypes of OSCC cells compared to the exon 15-deleted USO1 .

These observations suggest that LSM12 may function as a multifunctional protein that integrates RNA processing with calcium signaling pathways, potentially allowing for coordinated regulation of gene expression and cellular calcium dynamics. Further research is needed to directly investigate how these seemingly distinct functions might be mechanistically linked.

How does LSM12 expression vary across different types of neurons and glial cells?

• LSM12 at the RNA level is described as ubiquitous and abundantly expressed in different tissues and cell lines according to The Human Protein Atlas . This suggests that LSM12 is likely expressed in various neural cell types, though potentially at different levels.

• LSM12 has been implicated in neuroprotective pathways, particularly in the context of C9ORF72-associated amyotrophic lateral sclerosis (ALS) . This suggests functional relevance in motor neurons, which are primarily affected in ALS.

• The LSM12-EPAC1 axis defines a neuroprotective pathway that sustains the nucleocytoplasmic RAN gradient , which may be particularly important in neurons due to their polarized structure and compartmentalized signaling requirements.

• Given LSM12's role in NAADP-mediated calcium signaling and the importance of calcium signaling in neuronal function, it would be valuable to characterize LSM12 expression and function across different neuronal subtypes and glial cells.

Future research using single-cell RNA sequencing, immunohistochemistry on brain sections, and cell type-specific functional studies would help clarify the expression patterns and functions of LSM12 across the diversity of neural cell types.

What therapeutic applications might target LSM12 in cancer treatment?

Based on the research findings about LSM12's role in cancer, several potential therapeutic applications could be developed:

• LSM12 as a diagnostic biomarker: The dramatic upregulation of LSM12 in OSCC tissues (100% positive rate with 92% showing strong staining) compared to paired normal tissues (91% negative) suggests high sensitivity and specificity as a diagnostic marker . Similar expression patterns in other cancer types indicate potential broader applicability.

• RNA interference-based therapies: Since LSM12 knockdown significantly inhibited cancer cell growth, colony formation, migration, invasion, and tumor formation in vivo , the development of siRNA or shRNA therapeutics targeting LSM12 could be a promising approach.

• Small molecule inhibitors: Designing small molecules that disrupt LSM12's interaction with binding partners or that inhibit its splicing regulatory function could provide new therapeutic leads. For example, compounds that prevent LSM12-mediated inclusion of USO1 exon 15 might reduce cancer cell malignancy .

• Combination therapies: Understanding how LSM12 interacts with other cancer-associated pathways could reveal synergistic therapeutic targets. For instance, if LSM12's role in calcium signaling contributes to cancer progression, combining LSM12 inhibition with calcium signaling modulators might enhance therapeutic efficacy.

• Personalized medicine approaches: Stratifying patients based on LSM12 expression levels might help identify those most likely to benefit from LSM12-targeted therapies.

Future research should focus on validating these therapeutic concepts in preclinical models and exploring the potential side effects of targeting LSM12, given its expression in normal tissues and its role in fundamental cellular processes like calcium signaling.

How might LSM12 function be modulated for neuroprotection in neurodegenerative diseases?

Given LSM12's role in neuroprotective pathways, several approaches might be developed to modulate its function for therapeutic benefit in neurodegenerative diseases:

• Enhancing LSM12-EPAC1 pathway activity: Since the LSM12-EPAC1 axis defines a neuroprotective pathway that sustains the nucleocytoplasmic RAN gradient , pharmacological enhancement of this pathway might be beneficial in C9ORF72-associated ALS and potentially other neurodegenerative conditions.

• Targeting stress granule dynamics: LSM12 depletion increases poly(GR)-induced stress granules in models of C9ORF72-associated ALS . Developing compounds that mimic LSM12's effects on stress granule formation could help mitigate this pathological process.

• Modulating LSM12's interaction with ATXN2: Given the conserved biochemical association between LSM12 and ATXN2 , which is implicated in several neurodegenerative diseases, targeting this interaction could have therapeutic potential.

• Gene therapy approaches: In cases where LSM12 dysfunction contributes to disease, viral vector-mediated delivery of functional LSM12 to affected neural tissues might restore neuroprotective pathways.

• Using calcium signaling modulation: Since LSM12 functions as an NAADP receptor essential for NAADP-evoked calcium release , pharmacological tools that specifically modulate this pathway might provide neuroprotection in conditions where calcium dysregulation contributes to neurodegeneration.

These approaches would need to be carefully validated in relevant disease models before clinical translation, with particular attention to potential off-target effects given LSM12's diverse cellular functions.

What are the most critical unanswered questions about LSM12 biology that require further investigation?

Despite the significant advances in understanding LSM12 function, several critical questions remain unanswered:

• Structural biology of LSM12-NAADP interaction: What is the three-dimensional structure of LSM12, particularly its Lsm domain, and how does it specifically recognize and bind NAADP? Structural studies would provide insights for rational drug design.

• Regulation of LSM12 expression and activity: What factors control LSM12 expression in normal and pathological states? Are there post-translational modifications that regulate LSM12 function?

• Integration of LSM12's dual roles: How are LSM12's functions in RNA processing and calcium signaling integrated at the molecular and cellular levels? Does one function influence the other?

• Tissue-specific functions: Does LSM12 have tissue-specific roles or binding partners that explain its different implications in cancer versus neurodegeneration?

• Evolutionary conservation: How conserved is LSM12 function across species, and what can we learn from model organisms about its fundamental roles?

• Potential redundancy: Are there other proteins that can compensate for LSM12 function in its absence, particularly in the context of NAADP signaling?

• Diagnostic and therapeutic potential: Can LSM12 expression or activity be reliably measured in accessible patient samples for diagnostic purposes? What is the therapeutic window for targeting LSM12 in disease states?

• Connection to aging processes: Given LSM12's roles in stress responses and neurodegeneration, does it contribute to normal aging processes as suggested by some research?

Addressing these questions will require interdisciplinary approaches combining structural biology, biochemistry, cell biology, and translational research to fully understand LSM12's complex roles in health and disease.