AKT1 phosphorylates RBM17 to promote Sox2 transcription by modulating alternative splicing of FOXM1 to enhance cancer stem cell properties in colorectal cancer cells.

Colorectal cancer (CRC) is one of the leading causes of cancer-related death worldwide. The existence of cancer stem cells (CSC) causes tumor relapses, metastasis, and resistance to conventional therapy. Alternative splicing has been shown to affect physiological and pathological processes. Accumulating evidence has confirmed that targeting alternative splicing could be an effective strategy to treat CRC. Currently, the role of alternative splicing in the regulation of CSC properties in CRC has not been elucidated. Here, we show that RBM17 displays oncogenic roles in CRC cells. RBM17 enhances cell proliferation and reduces chemotherapeutic-induced apoptosis in CRC cells. Besides, RBM17 increases CD133 positive and ALDEFLUOR positive populations and promotes sphere formation in CRC cells. In mechanism studies, we found that FOXM1 is critical for RBM17 enhanced CSC properties. Moreover, FOXM1 alternative splicing is essential for RBM17 enhanced CSC properties in CRC cells. Additionally, RBM17 enhances CSC characteristics by controlling FOXM1 expression to promote Sox2 expression. Furthermore, AKT1 works as an upstream kinase to control RBM17-mediated FOXM1 alternative splicing and enhancement of CSC properties in CRC cells. Our study reveals that AKT1-RBM17-FOXM1-Sox2 axis could be a potential target for modulating alternative splicing to reduce CSC properties in CRC cells.

Selection and validation of novel stable reference genes for qPCR analysis in EMT and MET.

Quantitative real-time polymerase chain reaction is a powerful tool for quantifying gene expression. The relative quantification relies on normalizing the data to reference genes or internal controls not modulated by the experimental conditions. The most widely used internal controls occasionally show changed expression patterns in different experimental settings, such as the mesenchymal to epithelial transition. Thus, identifying appropriate internal controls is of utmost importance. We analyzed multiple RNA-Seq datasets using a combination of statistical approaches such as percent relative range and coefficient of variance to define a list of candidate internal control genes, which was then validated experimentally and by using in silico analyses as well. We identified a group of genes as strong internal control candidates with high stability compared to the classical ones. We also presented evidence for the superiority of the percent relative range method for calculating expression stability in data sets with larger sample sizes. We used multiple methods to analyze data collected from several RNA-Seq datasets; we identified Rbm17 and Katna1 as the most stable reference genes in EMT/MET studies. The percent relative range approach surpasses other methods when analyzing datasets of larger sample sizes.

RBM17 controls apoptosis and proliferation to promote Glioma progression.

The splicing factor SPF45 (RBM17) is a well-known component of the spliceosome that is involved in alternative splicing. RBM17 is frequently overexpressed in many tumors and plays a crucial role in cancer progression and drug resistance. However, the role of RBM17 in the development of glioma has not been thoroughly elucidated to date. In the present study, we found that RBM17 was overexpressed in glioma and that a high level of expression of RBM17 was closely associated with a poor prognosis in glioma patients. We investigated the effect of RBM17 on apoptosis, cell growth and cell cycle indexes and the activation of apoptosis signaling by shRNA in human U87 and U251 glioma cells. The downregulated expression of RBM17 mRNA was accompanied by the induction of cell cycle arrest, and apoptosis, reduced cell proliferation in the two cell lines, and reduced cell survival, as measured by the increased activation of caspase-3, caspase-9, and PARP (poly ADP-ribose polymerase). Furthermore, in subcutaneous U87 cell xenograft tumors in nude mice, intradermal administration of an shRNA targeting RBM17 significantly downregulated RBM17 expression in vivo and was accompanied by the suppressed growth of glioma. To the best of our knowledge, our results are the first to confirm that RBM17 functions in promoting cell proliferation, affecting the cell cycle, and inducing apoptosis in human glioma cells both in vitro and in vivo. These results indicate that RBM17 may be a therapeutic target in the clinical management of glioma.

Mechanism of Musashi2 affecting radiosensitivity of lung cancer by modulating DNA damage repair.

Identifying new targets for overcoming radioresistance is crucial for improving the efficacy of lung cancer radiotherapy, given that tumor cell resistance is a leading cause of treatment failure. Recent research has spotlighted the significance of Musashi2 (MSI2) in cancer biology. In this study, we first demonstrated that MSI2 plays a key function in regulating the radiosensitivity of lung cancer. The expression of MSI2 is negatively correlated with overall survival in cancer patients, and the knockdown of MSI2 inhibits tumorigenesis and increases radiosensitivity of lung cancer cells. Cellular radiosensitivity, which is closely linked to DNA damage, is influenced by MSI2 interaction with ataxia telangiectasia mutated and Rad3-related kinase (ATR) and checkpoint kinase 1 (CHK1) post-irradiation; moreover, knockdown of MSI2 inhibits the ATR-mediated DNA damage response pathway. RNA-binding motif protein 17 (RBM17), which is implicated in DNA damage repair, exhibits increased interaction with MSI2 post-irradiation. We found that knockdown of RBM17 disrupted the interaction between MSI2 and ATR post-irradiation and increased the radiosensitivity of lung cancer cells. Furthermore, we revealed the potential mechanism of MSI2 recruitment into the nucleus with the assistance of RBM17 to activate ATR to promote radioresistance. This study provides novel insights into the potential application of MSI2 as a new target in lung cancer radiotherapy.

RBM17 Expression Is Associated With the Efficacy of ICI Monotherapy in NSCLC With Low PD-L1 Expression.

BACKGROUND/AIM: Immune checkpoint inhibitors (ICIs) are currently a standard treatment tool for non-small cell lung cancer (NSCLC). RNA-binding motif protein 17 (RBM17), a splicing factor, is frequently over-expressed in NSCLC, but little is known about the role of RBM17 in the efficacy of ICIs for NSCLC. Thus, we investigated the correlation between RBM17 expression and ICI efficacy in NSCLC. PATIENTS AND METHODS: Biopsy or surgical specimens were collected from patients with advanced or recurrent NSCLC who received ICI monotherapy or chemo-immunotherapy in a first-line setting. RBM17 expression was examined using immunohistochemistry. The correlation between the efficacy of ICI monotherapy or chemo-immunotherapy and RBM17 expression was evaluated. RESULTS: Among the 218 cases, 115 (52.8%) cases were positive for RBM17 expression. RBM17 expression was not associated with the objective response rate (ORR) or progression-free survival (PFS) in either of the ICI monotherapy or chemo-immunotherapy groups. However, among those with a low PD-L1 expression level (PD-L1 <50%; n=86), RBM17 expression was significantly associated with a better ORR (p=0.045) and a better PFS (p<0.001) in the ICI monotherapy group, and was significantly associated with a poor ORR in the chemo-immunotherapy group (p=0.041). CONCLUSION: RBM17 might be a useful predictive marker for a higher efficacy of ICI monotherapy in NSCLC patients with a low PD-L1 expression level.

SAP30BP interacts with RBM17/SPF45 to promote splicing in a subset of human short introns.

Human pre-mRNA splicing requires the removal of introns with highly variable lengths, from tens to over a million nucleotides. Therefore, mechanisms of intron recognition and splicing are likely not universal. Recently, we reported that splicing in a subset of human short introns with truncated polypyrimidine tracts depends on RBM17 (SPF45), instead of the canonical splicing factor U2 auxiliary factor (U2AF) heterodimer. Here, we demonstrate that SAP30BP, a factor previously implicated in transcriptional control, is an essential splicing cofactor for RBM17. In vitro binding and nuclear magnetic resonance analyses demonstrate that a U2AF-homology motif (UHM) in RBM17 binds directly to a newly identified UHM-ligand motif in SAP30BP. We show that this RBM17-SAP30BP interaction is required to specifically recruit RBM17 to phosphorylated SF3B1 (SF3b155), a U2 small nuclear ribonucleoprotein (U2 snRNP) component in active spliceosomes. We propose a mechanism for splicing in a subset of short introns, in which SAP30BP guides RBM17 in the assembly of active spliceosomes.

A 5'-tRNA halve, tiRNA-Gly promotes cell proliferation and migration via binding to RBM17 and inducing alternative splicing in papillary thyroid cancer.

BACKGROUND: tRNA-derived small noncoding RNAs (sncRNAs) are mainly categorized into tRNA halves (tiRNAs) and fragments (tRFs). Biological functions of tiRNAs in human solid tumor are attracting more and more attention, but researches concerning the mechanisms in tiRNAs-mediated tumorigenesis are rarely. The direct regulatory relationship between tiRNAs and splicing-related proteins remain elusive. METHODS: Papillary thyroid carcinoma (PTC) associated tRNA fragments were screened by tRNA fragments deep sequencing and validated by qRT-PCR and Northern Blot in PTC tissues. The biological function of tRNA fragments were assessed by cell counting kit, transwells and subcutaneous transplantation tumor of nude mice. For mechanistic study, tRNA fragments pull-down, RNA immunoprecipitation, Western Blot, Immunofluorescence, Immunohistochemical staining were performed. RESULTS: Herein, we have identified a 33 nt tiRNA-Gly significantly increases in papillary thyroid cancer (PTC) based on tRFs & tiRNAs sequencing. The ectopic expression of tiRNA-Gly promotes cell proliferation and migration, whereas down-regulation of tiRNA-Gly exhibits reverse effects. Mechanistic investigations reveal tiRNA-Gly directly bind the UHM domain of a splicing-related RNA-binding protein RBM17. The interaction with tiRNA-Gly could translocate RBM17 from cytoplasm into nucleus. In addition, tiRNA-Gly increases RBM17 protein expression via inhibiting its degradation in a ubiquitin/proteasome-dependent way. Moreover, RBM17 level in tiRNA-Gly high-expressing human PTC tissues is upregulated. In vivo mouse model shows that suppression of tiRNA-Gly decreases RBM17 expression. Importantly, tiRNA-Gly can induce exon 16 splicing of MAP4K4 mRNA leading to phosphorylation of downstream signaling pathway, which is RBM17 dependent. CONCLUSIONS: Our study firstly illustrates tiRNA-Gly can directly bind to RBM17 and display oncogenic effect via RBM17-mediated alternative splicing. This fully novel model broadens our understanding of molecular mechanism in which tRNA fragment in tumor cells directly bind RNA binding protein and play a role in alternative splicing.

SPF45/RBM17-dependent splicing and multidrug resistance to cancer chemotherapy.

The early splicing complex A occupies at least eighty nucleotides of intron, in which U2AF covers the polypyrimidine tract. SPF45 (RBM17) functionally substitutes for U2AF on a subset of short introns. Since SPF45 expression confers resistance to various anticancer drugs, SPF45-dependent splicing may play a critical role in multidrug resistance.

RBM17 Interacts with U2SURP and CHERP to Regulate Expression and Splicing of RNA-Processing Proteins.

RNA splicing entails the coordinated interaction of more than 150 proteins in the spliceosome, one of the most complex of the cell's molecular machines. We previously discovered that the RNA-binding motif protein 17 (RBM17), a component of the spliceosome, is essential for survival and cell maintenance. Here, we find that it interacts with the spliceosomal factors U2SURP and CHERP and that they reciprocally regulate each other's stability, both in mouse and in human cells. Individual knockdown of each of the three proteins induces overlapping changes in splicing and gene expression of transcripts enriched for RNA-processing factors. Our results elucidate the function of RBM17, U2SURP, and CHERP and link the activity of the spliceosome to the regulation of downstream RNA-binding proteins. These data support the hypothesis that, beyond driving constitutive splicing, spliceosomal factors can regulate alternative splicing of specific targets.