Novel HNRNPM::LEUTX fusion resulting from chromothripsis of chromosome 19 in a pediatric undifferentiated small round cell neoplasm.

Small round cell neoplasms comprise a diverse group of tumors characterized by a primitive/undifferentiated appearance. Although several entities are associated with recurrent gene fusions, many of these neoplasms have not been fully characterized, and novel molecular alterations are being discovered. Here, we report an undifferentiated small round cell neoplasm arising in the anterior mediastinum of a 17-month-old female. The tumor harbored a novel HNRNPM::LEUTX fusion resulting from chromothripsis of chromosome 19, which was identified by whole transcriptome sequencing, but not by targeted sequencing. The structural variations caused by the chromothripsis event also challenged the interpretation of the targeted sequencing findings. This report expands the spectrum of gene partners involved in LEUTX fusions and underscores the value of whole transcriptome sequencing in the diagnostic workup of undifferentiated small round cell tumors. It also highlights the interpretive challenges associated with complex genomic alterations. A careful evidence-based analysis of sequencing data along with histopathologic correlation is essential to ensure correct categorization of fusions.

A short report of novel RARG-HNRNPM fusion gene in resembling acute promyelocytic leukemia.

BACKGROUND: Resembling acute promyelocytic leukemia (APL) is a unique subtype of APL who sharing clinical, morphological, and immunophenotypic features with typical APL, but lacking evidence of PML-RARA fusion gene and usually insensitive to arsenic trioxide (ATO) and all-trans retinoic acid (ATRA). For years, RARA, RARB and RARG rearrangement were found in resembling APL continually. The confirmed partner genes of RARG rearrangement included CPSF6, NUP98, NPM1, PML, and HNRNPC. These patients were a group of resembling APL with rare molecular genetic abnormality and unfavorable prognosis. They usually were resistant to ATO and ATRA but partially sensitive to anthracycline-based chemotherapy. CASE PRESENTATION: We reported a 25-year-old female patient with a novel fusion gene RARG-HNRNPM (RARG chr12:53606869: -; HNRNPM chr19: 8527413: + based on GRCh37/hg19 Assembly) through RNA-seq as resembling APL. The patient with RARG-HNRNPM was benefited from a combined chemotherapy homoharringtonine, cytarabine, and aclacinomycin (HAA) regimen with no relapse. DISCUSSION AND CONCLUSIONS: RARG rearrangement resembling APL are various. The treatment should be switched from ATRA/ATO to AML combined chemotherapy regimen early.

CircURI1 interacts with hnRNPM to inhibit metastasis by modulating alternative splicing in gastric cancer.

Circular RNAs (circRNAs) have emerged as key regulators of human cancers, yet their modes of action in gastric cancer (GC) remain largely unknown. Here, we identified circURI1 back-spliced from exons 3 and 4 of unconventional prefoldin RPB5 interactor 1 (URI1) from circRNA profiling of five-paired human gastric and the corresponding nontumor adjacent specimens (paraGC). CircURI1 exhibits the significantly higher expression in GC compared with paraGC and inhibitory effects on cell migration and invasion in vitro and GC metastasis in vivo. Mechanistically, circURI1 directly interacts with heterogeneous nuclear ribonucleoprotein M (hnRNPM) to modulate alternative splicing of genes, involved in the process of cell migration, thus suppressing GC metastasis. Collectively, our study expands the current knowledge regarding the molecular mechanism of circRNA-mediated cancer metastasis via modulating alternative splicing.

The pan-cancer lncRNA PLANE regulates an alternative splicing program to promote cancer pathogenesis.

Genomic amplification of the distal portion of chromosome 3q, which encodes a number of oncogenic proteins, is one of the most frequent chromosomal abnormalities in malignancy. Here we functionally characterise a non-protein product of the 3q region, the long noncoding RNA (lncRNA) PLANE, which is upregulated in diverse cancer types through copy number gain as well as E2F1-mediated transcriptional activation. PLANE forms an RNA-RNA duplex with the nuclear receptor co-repressor 2 (NCOR2) pre-mRNA at intron 45, binds to heterogeneous ribonucleoprotein M (hnRNPM) and facilitates the association of hnRNPM with the intron, thus leading to repression of the alternative splicing (AS) event generating NCOR2-202, a major protein-coding NCOR2 AS variant. This is, at least in part, responsible for PLANE-mediated promotion of cancer cell proliferation and tumorigenicity. These results uncover the function and regulation of PLANE and suggest that PLANE may constitute a therapeutic target in the pan-cancer context.

HNRNPM controls circRNA biogenesis and splicing fidelity to sustain cancer cell fitness.

High spliceosome activity is a dependency for cancer cells, making them more vulnerable to perturbation of the splicing machinery compared to normal cells. To identify splicing factors important for prostate cancer (PCa) fitness, we performed pooled shRNA screens in vitro and in vivo. Our screens identified heterogeneous nuclear ribonucleoprotein M (HNRNPM) as a regulator of PCa cell growth. RNA- and eCLIP-sequencing identified HNRNPM binding to transcripts of key homeostatic genes. HNRNPM binding to its targets prevents aberrant exon inclusion and backsplicing events. In both linear and circular mis-spliced transcripts, HNRNPM preferentially binds to GU-rich elements in long flanking proximal introns. Mimicry of HNRNPM-dependent linear-splicing events using splice-switching-antisense-oligonucleotides was sufficient to inhibit PCa cell growth. This suggests that PCa dependence on HNRNPM is likely a result of mis-splicing of key homeostatic coding and non-coding genes. Our results have further been confirmed in other solid tumors. Taken together, our data reveal a role for HNRNPM in supporting cancer cell fitness. Inhibition of HNRNPM activity is therefore a potential therapeutic strategy in suppressing growth of PCa and other solid tumors.

hnRNPM deficiency leads to cognitive deficits via disrupting synaptic plasticity.

RNA metabolism involves complex and regulated processes, some of which include transcription, intracellular transport, translation, and degradation. The involvement of RNA binding proteins in these processes remains mostly uncharacterized regarding brain functions, especially cognition. In this study, we report that knockdown of hnRNPM in the CA1 hippocampal region of the mouse brain leads to learning and memory impairment. This finding is further supported, by the reduction of pre- and post-synaptic protein levels synaptophysin and PSD95. Notably, loss of hnRNPM affects the physiological spine in vivo by impairing the morphology of the dendritic spines. Additionally, our study demonstrates that hnRNPM directly binds to the 3'UTR of synaptophysin and PSD95 mRNAs, resulting in the stabilization of these mRNAs. Together, these findings present novel insight into the regulatory role of hnRNPM in neuronal structure and function.

Discovery of anti-cell migration activity of an anti-HIV heterocyclic compound by identification of its binding protein hnRNP M.

One compound sometimes shows two biological functions, becoming important aspect of recent drug discovery. This study began with an attempt to confirm the previously reported molecular mechanism of the anti-human immunodeficiency virus (HIV) heterocyclic compound BMMP [2-(benzothiazol-2-ylmethylthio)-4-methylpyrimidine], i.e., induction of abnormal uncoating of the viral core at the post-entry step. Our mechanistic study gave results consistent with this mechanism. We further attempted to find out the molecular target of BMMP by a pulldown approach using previously synthesized biotinylated BMMP (Biotin-BMMP) and successfully identified heterogenous nuclear ribonucleoprotein M (hnRNP M) as a BMMP-binding protein. This protein was found not to be accountable for the anti-HIV activity of BMMP. As hnRNP M has been reported to promote cancer metastasis, we tested this mechanism and found that BMMP suppressed migration of the human lung carcinoma cell line A549 stimulated with transforming growth factor-ß (TGF-ß). Mechanistic study showed that BMMP suppressed the expression of CD44 mRNA via the regulation of hnRNP M. Furthermore, six new derivatives of BMMP were synthesized, and the patterns of their activities against HIV-1 and cell migration were not uniform, suggesting that the anti-HIV mechanism and the anti-cell migration mechanism of BMMP are independent. Taken together, the anti-cell migration activity of the anti-HIV heterocyclic compound BMMP was newly discovered by identification of its binding protein hnRNP M using a chemical biology approach.

The RNA-binding protein AKAP8 suppresses tumor metastasis by antagonizing EMT-associated alternative splicing.

Alternative splicing has been shown to causally contribute to the epithelial-mesenchymal transition (EMT) and tumor metastasis. However, the scope of splicing factors that govern alternative splicing in these processes remains largely unexplored. Here we report the identification of A-Kinase Anchor Protein (AKAP8) as a splicing regulatory factor that impedes EMT and breast cancer metastasis. AKAP8 not only is capable of inhibiting splicing activity of the EMT-promoting splicing regulator hnRNPM through protein-protein interaction, it also directly binds to RNA and alters splicing outcomes. Genome-wide analysis shows that AKAP8 promotes an epithelial cell state splicing program. Experimental manipulation of an AKAP8 splicing target CLSTN1 revealed that splice isoform switching of CLSTN1 is crucial for EMT. Moreover, AKAP8 expression and the alternative splicing of CLSTN1 predict breast cancer patient survival. Together, our work demonstrates the essentiality of RNA metabolism that impinges on metastatic breast cancer.

The Splicing Factor hnRNP M Is a Critical Regulator of Innate Immune Gene Expression in Macrophages.

While transcriptional control of innate immune gene expression is well characterized, almost nothing is known about how pre-mRNA splicing decisions influence, or are influenced by, macrophage activation. Here, we demonstrate that the splicing factor hnRNP M is a critical repressor of innate immune gene expression and that its function is regulated by pathogen sensing cascades. Loss of hnRNP M led to hyperinduction of a unique regulon of inflammatory and antimicrobial genes following diverse innate immune stimuli. While mutating specific serines on hnRNP M had little effect on its ability to control pre-mRNA splicing or transcript levels of housekeeping genes in resting macrophages, it greatly impacted the protein's ability to dampen induction of specific innate immune transcripts following pathogen sensing. These data reveal a previously unappreciated role for pattern recognition receptor signaling in controlling splicing factor phosphorylation and establish pre-mRNA splicing as a critical regulatory node in defining innate immune outcomes.

The heterogeneous nuclear ribonucleoprotein hnRNPM inhibits RNA virus-triggered innate immunity by antagonizing RNA sensing of RIG-I-like receptors.

Recognition of viral RNA by the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), including RIG-I and MDA5, initiates innate antiviral responses. Although regulation of RLR-mediated signal transduction has been extensively investigated, how the recognition of viral RNA by RLRs is regulated remains enigmatic. In this study, we identified heterogeneous nuclear ribonucleoprotein M (hnRNPM) as a negative regulator of RLR-mediated signaling. Overexpression of hnRNPM markedly inhibited RNA virus-triggered innate immune responses. Conversely, hnRNPM-deficiency increased viral RNA-triggered innate immune responses and inhibited replication of RNA viruses. Viral infection caused translocation of hnRNPM from the nucleus to the cytoplasm. hnRNPM interacted with RIG-I and MDA5, and impaired the binding of the RLRs to viral RNA, leading to inhibition of innate antiviral response. Our findings suggest that hnRNPM acts as an important decoy for excessive innate antiviral immune response.

hnRNPM, a potential mediator of YY1 in promoting the epithelial-mesenchymal transition of prostate cancer cells.

BACKGROUND: With the popularity of serum prostate-specific antigen (PSA) screening, the number of newly diagnosed prostate cancer (PCa) patients is increasing. However, indolent or invasive PCa cannot be distinguished by PSA levels. Here, we mainly explored the role of heterogeneous nuclear ribonucleoprotein M (hnRNPM) in the invasiveness of PCa. METHODS: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis was used to detect the expressions of hnRNPM in PCa and benign prostate hyperplasia (BPH) tissues as well as in PCa cell lines. Immunohistochemistry was applied to detect the hnRNPM or Yin Yang 1 (YY1) expression in BPH, prostate adenocarcinoma (ADENO) and neuroendocrine prostate cancer (NEPC) tissues. After aberrant, the expression of hnRNPM in C4-2 and PC3 cells, the changes of cell migration and invasion were observed through wound-healing and transwell assays. We also predicted the transcription factor of hnRNPM through databases, then verified the association of hnRNPM and YY1 using chromatin immunoprecipitation (ChIP) and luciferase assays. RESULTS: The expression level of hnRNPM is gradually reduced in BPH, ADENO, and NEPC tissues and it is less expressed in more aggressive PCa cell lines. Overexpression of hnRNPM can significantly reduce Twist1 expression, which inhibits the migration and invasion of PCa cells in vitro. In PCa cells, overexpression of YY1 can promote epithelial-mesenchymal transition by reducing hnRNPM expression. Furthermore, this effect caused by overexpression of YY1 can be partially attenuated by simultaneous overexpression of hnRNPM. CONCLUSIONS: Our study demonstrates that hnRNPM negatively regulated PCa cell migration and invasion, and its expression can be transcriptionally inhibited by YY1. We speculated that hnRNPM may be a biomarker to assist in judging the aggressiveness of PCa.

hnRNPM induces translation switch under hypoxia to promote colon cancer development.

BACKGROUND: Hypoxia suppresses global protein production, yet certain essential proteins are translated through alternative pathways to survive under hypoxic stress. Translation via the internal ribosome entry site (IRES) is a means to produce proteins under stress conditions such as hypoxia; however, the underlying mechanism remains largely uncharacterized. METHODS: Proteomic and bioinformatic analyses were employed to identify hnRNPM as an IRES interacting factor. Clinical specimens and mouse model of tumorigenesis were used for determining the expression and correlation of hnRNPM and its target gene. Transcriptomic and translatomic analyses were performed to profile target genes regulated by hnRNPM. FINDINGS: Hypoxia increases cytosolic hnRNPM binding onto its target mRNAs and promotes translation initiation. Clinical colon cancer specimens and mouse carcinogenesis model showed that hnRNPM is elevated during the development of colorectal cancer, and is associated with poor prognosis. Genome-wide transcriptomics and translatomics analyses revealed a unique set of hnRNPM-targeted genes involved in metabolic processes and cancer neoplasia are selectively translated under hypoxia. INTERPRETATION: These data highlight the critical role of hnRNPM-IRES-mediated translation in transforming hypoxia-induced proteome toward malignancy. FUND: This work was supported by the Ministry of Science and Technology, Taiwan (MOST 104-2320-B-006-042 to HSS and MOST 105-2628-B-001-MY3 to TMC).

HnRNPM and CD44s expression affects tumor aggressiveness and predicts poor prognosis in breast cancer with axillary lymph node metastases.

HnRNPM is an essential splicing factor and its expression is closely correlated with invasion and metastasis of tumor cells. The CD44 cell adhesion molecule is aberrantly expressed in many breast tumors and CD44 splice variants have been implicated in specific oncogenic signaling pathways. To investigate the clinical significance and biological function of hnRNPM, immunohistochemistry, quantitative, and semiquantitative polymerase chain reaction, lentiviral transfection system and transwell invasion assays were performed. We found that hnRNPM expression was significantly upregulated in breast cancer tissues compared with benign breast lesions. Although there was no significant correlation between hnRNPM and total CD44 protein or mRNA level, there was a negative correlation between hnRNPM and CD44v6. HnRNPM and CD44s expression showed positive correlation and in particular, they were dually expressed in breast cancer tissues. Interestingly, cancer stem cells marker, ALDH1+ phenotype was positively associated with overexpression of CD44s or hnRNPM and negatively related to CD44v6. Patients with high hnRNPM tended to have higher levels of CD44s, shorter overall survival (OS) and higher rates of lymph node metastases (LNM). Remarkably, Kaplan-Meier and Cox regression analyses displayed that hnRNPM+ or CD44shigh was a poor prognostic factor for OS of patients with LNM. Upregulation of hnRNPM in MCF-7 cells caused a significant increase in cell invasion, and this effect may occur through the regulation of CD44s expression. In conclusion, overexpression of hnRNPM promotes breast cancer aggressiveness by regulating the level of CD44s, indicates a poor prognosis for patients with LNM, and has potential as therapeutic targets.

Cell type-restricted activity of hnRNPM promotes breast cancer metastasis via regulating alternative splicing.

Tumor metastasis remains the major cause of cancer-related death, but its molecular basis is still not well understood. Here we uncovered a splicing-mediated pathway that is essential for breast cancer metastasis. We show that the RNA-binding protein heterogeneous nuclear ribonucleoprotein M (hnRNPM) promotes breast cancer metastasis by activating the switch of alternative splicing that occurs during epithelial-mesenchymal transition (EMT). Genome-wide deep sequencing analysis suggests that hnRNPM potentiates TGFß signaling and identifies CD44 as a key downstream target of hnRNPM. hnRNPM ablation prevents TGFß-induced EMT and inhibits breast cancer metastasis in mice, whereas enforced expression of the specific CD44 standard (CD44s) splice isoform overrides the loss of hnRNPM and permits EMT and metastasis. Mechanistically, we demonstrate that the ubiquitously expressed hnRNPM acts in a mesenchymal-specific manner to precisely control CD44 splice isoform switching during EMT. This restricted cell-type activity of hnRNPM is achieved by competition with ESRP1, an epithelial splicing regulator that binds to the same cis-regulatory RNA elements as hnRNPM and is repressed during EMT. Importantly, hnRNPM is associated with aggressive breast cancer and correlates with increased CD44s in patient specimens. These findings demonstrate a novel molecular mechanism through which tumor metastasis is endowed by the hnRNPM-mediated splicing program.

Identification of HnRNP M as a novel biomarker for colorectal carcinoma by quantitative proteomics.

Colorectal carcinoma (CRC) is one of the most common cancers in the world, and identification of new CRC biomarkers will be helpful for the diagnosis and treatment of CRC. For isobaric tags for relative and absolute quantitation (iTRAQ) analysis, fresh CRC and adjacent, colonic adenoma, ulcerative colitis, Crohn's disease, and noncancerous colonic epithelial tissue were obtained from patients at the 2nd Xiangya Hospital of Central South University, China. The function of heterogeneous nuclear ribonucleoprotein M (HnRNP M) during the proliferation, invasion, and metastasis of CRC cells in vitro was evaluated. One hundred and twenty-six differentially expressed proteins were identified by iTRAQ analysis. The expression of HnRNP M exhibited progressive changes during the carcinogenic process and was validated by Western blot. The upregulation of HnRNP M correlated with cancer recurrence and regional lymph node metastasis. Furthermore, biological role exploration suggests that HnRNP M positively regulates cell cycle progression, promotes cell growth and invasion in vitro, and increases the colony-forming ability of LS174T cells. The present data demonstrate that the upregulation of HnRNP M is involved in human colorectal epithelial carcinogenesis and may serve as a carcinoma biomarker for CRC.

Regulatory roles of heterogeneous nuclear ribonucleoprotein M and Nova-1 protein in alternative splicing of dopamine D2 receptor pre-mRNA.

The dopamine D2 receptor (D2R) plays a crucial role in the regulation of diverse key physiological functions, including motor control, reward, learning, and memory. This receptor is present in vivo in two isoforms, D2L and D2S, generated from the same gene by alternative pre-mRNA splicing. Each isoform has a specific role in vivo, underlining the importance of a strict control of its synthesis, yet the molecular mechanism modulating alternative D2R pre-mRNA splicing has not been completely elucidated. Here, we identify heterogeneous nuclear ribonucleoprotein M (hnRNP M) as a key molecule controlling D2R splicing. We show that binding of hnRNP M to exon 6 inhibited the inclusion of this exon in the mRNA. Importantly, the splicing factor Nova-1 counteracted hnRNP M effects on D2R pre-mRNA splicing. Indeed, mutations of the putative Nova-1-binding site on exon 6 disrupted Nova-1 RNA assembly and diminished the inhibitory effect of Nova-1 on hnRNP M-dependent exon 6 exclusion. These results identify Nova-1 and hnRNP M as D2R pre-mRNA-binding proteins and show their antagonistic role in the alternative splicing of D2R pre-mRNA.

Mechanistic control of carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) splice isoforms by the heterogeneous nuclear ribonuclear proteins hnRNP L, hnRNP A1, and hnRNP M.

Carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) is expressed in a variety of cell types and is implicated in carcinogenesis. Alternative splicing of CEACAM1 pre-mRNA generates two cytoplasmic domain splice variants characterized by the inclusion (L-isoform) or exclusion (S-isoform) of exon 7. Here we show that the alternative splicing of CEACAM1 pre-mRNA is regulated by novel cis elements residing in exon 7. We report the presence of three exon regulatory elements that lead to the inclusion or exclusion of exon 7 CEACAM1 mRNA in ZR75 breast cancer cells. Heterologous splicing reporter assays demonstrated that the maintenance of authentic alternative splicing mechanisms were independent of the CEACAM1 intron sequence context. We show that forced expression of these exon regulatory elements could alter CEACAM1 splicing in HEK-293 cells. Using RNA affinity chromatography, three members of the heterogeneous nuclear ribonucleoprotein family (hnRNP L, hnRNP A1, and hnRNP M) were identified. RNA immunoprecipitation of hnRNP L and hnRNP A1 revealed a binding motif located central and 3' to exon 7, respectively. Depletion of hnRNP A1 or L by RNAi in HEK-293 cells promoted exon 7 inclusion, whereas overexpression led to exclusion of the variable exon. By contrast, overexpression of hnRNP M showed exon 7 inclusion and production of CEACAM1-L mRNA. Finally, stress-induced cytoplasmic accumulation of hnRNP A1 in MDA-MB-468 cells dynamically alters the CEACAM1-S:CEACAM1:L ratio in favor of the l-isoform. Thus, we have elucidated the molecular factors that control the mechanism of splice-site recognition in the alternative splicing regulation of CEACAM1.