Identification of long non-coding RNAs and RNA binding proteins in breast cancer subtypes.

Breast cancer is a heterogeneous disease classified into four main subtypes with different clinical outcomes, such as patient survival, prognosis, and relapse. Current genetic tests for the differential diagnosis of BC subtypes showed a poor reproducibility. Therefore, an early and correct diagnosis of molecular subtypes is one of the challenges in the clinic. In the present study, we identified differentially expressed genes, long non-coding RNAs and RNA binding proteins for each BC subtype from a public dataset applying bioinformatics algorithms. In addition, we investigated their interactions and we proposed interacting biomarkers as potential signature specific for each BC subtype. We found a network of only 2 RBPs (RBM20 and PCDH20) and 2 genes (HOXB3 and RASSF7) for luminal A, a network of 21 RBPs and 53 genes for luminal B, a HER2-specific network of 14 RBPs and 30 genes, and a network of 54 RBPs and 302 genes for basal BC. We validated the signature considering their expression levels on an independent dataset evaluating their ability to classify the different molecular subtypes with a machine learning approach. Overall, we achieved good performances of classification with an accuracy >0.80. In addition, we found some interesting novel prognostic biomarkers such as RASSF7 for luminal A, DCTPP1 for luminal B, DHRS11, KLC3, NAGS, and TMEM98 for HER2, and ABHD14A and ADSSL1 for basal. The findings could provide preliminary evidence to identify putative new prognostic biomarkers and therapeutic targets for individual breast cancer subtypes.

The protective role of m1A during stress-induced granulation.

Post-transcriptional methylation of N6-adenine and N1-adenine can affect transcriptome turnover and translation. Furthermore, the regulatory function of N6-methyladenine (m6A) during heat shock has been uncovered, including the enhancement of the phase separation potential of RNAs. In response to acute stress, e.g. heat shock, the orderly sequestration of mRNAs in stress granules (SGs) is considered important to protect transcripts from the irreversible aggregation. Until recently, the role of N1-methyladenine (m1A) on mRNAs during acute stress response remains largely unknown. Here we show that the methyltransferase complex TRMT6/61A, which generates the m1A tag, is involved in transcriptome protection during heat shock. Our bioinformatics analysis indicates that occurrence of the m1A motif is increased in mRNAs known to be enriched in SGs. Accordingly, the m1A-generating methyltransferase TRMT6/61A accumulated in SGs and mass spectrometry confirmed enrichment of m1A in the SG RNAs. The insertion of a single methylation motif in the untranslated region of a reporter RNA leads to more efficient recovery of protein synthesis from that transcript after the return to normal temperature. Our results demonstrate far-reaching functional consequences of a minimal RNA modification on N1-adenine during acute proteostasis stress.

Posttranscriptional regulation of colonic epithelial repair by RNA binding protein IMP1/IGF2BP1.

RNA binding proteins, including IMP1/IGF2BP1, are essential regulators of intestinal development and cancer. Imp1 hypomorphic mice exhibit gastrointestinal growth defects, yet the specific role for IMP1 in colon epithelial repair is unclear. Our prior work revealed that intestinal epithelial cell-specific Imp1 deletion (Imp1ΔIEC ) was associated with better regeneration in mice after irradiation. Here, we report increased IMP1 expression in patients with Crohn's disease and ulcerative colitis. We demonstrate that Imp1ΔIEC mice exhibit enhanced recovery following dextran sodium sulfate (DSS)-mediated colonic injury. Imp1ΔIEC mice exhibit Paneth cell granule changes, increased autophagy flux, and upregulation of Atg5. In silico and biochemical analyses revealed direct binding of IMP1 to MAP1LC3B, ATG3, and ATG5 transcripts. Genetic deletion of essential autophagy gene Atg7 in Imp1ΔIEC mice revealed increased sensitivity of double-mutant mice to colonic injury compared to control or Atg7 single mutant mice, suggesting a compensatory relationship between Imp1 and the autophagy pathway. The present study defines a novel interplay between IMP1 and autophagy, where IMP1 may be transiently induced during damage to modulate colonic epithelial cell responses to damage.

The moonlighting RNA-binding activity of cytosolic serine hydroxymethyltransferase contributes to control compartmentalization of serine metabolism.

Enzymes of intermediary metabolism are often reported to have moonlighting functions as RNA-binding proteins and have regulatory roles beyond their primary activities. Human serine hydroxymethyltransferase (SHMT) is essential for the one-carbon metabolism, which sustains growth and proliferation in normal and tumour cells. Here, we characterize the RNA-binding function of cytosolic SHMT (SHMT1) in vitro and using cancer cell models. We show that SHMT1 controls the expression of its mitochondrial counterpart (SHMT2) by binding to the 5'untranslated region of the SHMT2 transcript (UTR2). Importantly, binding to RNA is modulated by metabolites in vitro and the formation of the SHMT1-UTR2 complex inhibits the serine cleavage activity of the SHMT1, without affecting the reverse reaction. Transfection of UTR2 in cancer cells controls SHMT1 activity and reduces cell viability. We propose a novel mechanism of SHMT regulation, which interconnects RNA and metabolites levels to control the cross-talk between cytosolic and mitochondrial compartments of serine metabolism.

Protein Syndesmos is a novel RNA-binding protein that regulates primary cilia formation.

Syndesmos (SDOS) is a functionally poorly characterized protein that directly interacts with p53 binding protein 1 (53BP1) and regulates its recruitment to chromatin. We show here that SDOS interacts with another important cancer-linked protein, the chaperone TRAP1, associates with actively translating polyribosomes and represses translation. Moreover, we demonstrate that SDOS directly binds RNA in living cells. Combining individual gene expression profiling, nucleotide crosslinking and immunoprecipitation (iCLIP), and ribosome profiling, we discover several crucial pathways regulated post-transcriptionally by SDOS. Among them, we identify a small subset of mRNAs responsible for the biogenesis of primary cilium that have been linked to developmental and degenerative diseases, known as ciliopathies, and cancer. We discover that SDOS binds and regulates the translation of several of these mRNAs, controlling cilia development.

FUS Phase Separation Is Modulated by a Molecular Chaperone and Methylation of Arginine Cation-π Interactions.

Reversible phase separation underpins the role of FUS in ribonucleoprotein granules and other membrane-free organelles and is, in part, driven by the intrinsically disordered low-complexity (LC) domain of FUS. Here, we report that cooperative cation-π interactions between tyrosines in the LC domain and arginines in structured C-terminal domains also contribute to phase separation. These interactions are modulated by post-translational arginine methylation, wherein arginine hypomethylation strongly promotes phase separation and gelation. Indeed, significant hypomethylation, which occurs in FUS-associated frontotemporal lobar degeneration (FTLD), induces FUS condensation into stable intermolecular ß-sheet-rich hydrogels that disrupt RNP granule function and impair new protein synthesis in neuron terminals. We show that transportin acts as a physiological molecular chaperone of FUS in neuron terminals, reducing phase separation and gelation of methylated and hypomethylated FUS and rescuing protein synthesis. These results demonstrate how FUS condensation is physiologically regulated and how perturbations in these mechanisms can lead to disease.