BC200 is a long non-coding RNA primarily expressed in brain but aberrantly expressed in various cancers. To gain a further understanding of the function of BC200, we performed proteomic analyses of the BC200 ribonucleoprotein (RNP) by transfection of 3′ DIG-labelled BC200. Protein binding partners of the functionally related murine RNA BC1 as well as a scrambled BC200 RNA were also assessed in both human and mouse cell lines. Stringent validation of proteins identified by mass spectrometry confirmed 14 of 84 protein binding partners and excluded eight proteins that did not appreciably bind BC200 in reverse experiments. Gene ontology analyses revealed general roles in RNA metabolic processes, RNA processing and splicing. Protein/RNA interaction sites were mapped with a series of RNA truncations revealing three distinct modes of interaction involving either the 5′ Alu-domain, 3′ A-rich or 3′ C-rich regions. Due to their high enrichment values in reverse experiments, CSDE1 and STRAP were further analyzed demonstrating a direct interaction between CSDE1 and BC200 and indirect binding of STRAP to BC200 via heterodimerization with CSDE1. Knock-down studies identified a reciprocal regulatory relationship between CSDE1 and BC200 and immunofluorescence analysis of BC200 knock-down cells demonstrated a dramatic reorganization of CSDE1 into distinct nuclear foci.
BC200 is a non-coding RNA elevated in a broad spectrum of tumour cells that is critical for cell viability, invasion, and migration. Over-expression studies have implicated BC200 and the rodent analog BC1 as negative regulators of translation in both cell-based and in vitro translation assays. While consistent, these studies have not been confirmed in knock-down studies and direct evidence for this function is lacking. Herein, we have demonstrated that BC200 knock-down is correlated with a decrease in global translation rates. As this conflicts with the hypothesis that BC200 is a translational suppressor, we overexpressed BC200 by transfection of in vitro transcribed RNA and transient expression from transfected plasmids. In this context BC200 suppressed translation; however, an innate immune response confounded the data. To overcome this, breast cancer cells stably overexpressing BC200 and various control RNAs were developed by selection for genomic incorporation of a plasmid co-expressing BC200 and the neomycin resistance gene. Stable overexpression of BC200 was associated with elevated translation levels in pooled stable cell lines and isolated single-cell clones. Crosslinking sucrose density gradient centrifugation demonstrated an association of BC200 and its reported binding partners SRP9/14, CSDE1, DHX36 and PABPC1 with both ribosomal subunits and polysomal RNA, an association not previously observed due to the labile nature of the interactions. In summary, these data present a novel understanding of BC200 function as well as optimized methodology that has far reaching implications in the study of non-coding RNAs, particularly within the context of translational regulatory mechanisms.
BACKGROUND Klippel-Trenaunay-Weber syndrome (KTWS) is a very rare syndrome that involves three conditions: Cutaneous hemangiomas, varicosities, and soft-tissue hypertrophy of the affected limb. There are few cases of ischemic infarction with KTWS. Here, we describe a case of KTWS with ischemic stroke. CASE SUMMARY A 43-year-old man was diagnosed with KTWS with ischemic stroke. His chief complaints were worsening weakness and spasticity in the right leg. These symptoms had been present for 1 year, but the patient did not receive comprehensive rehabilitation until he underwent a 3-week integrated inpatient rehabilitation program at our center. After the program, his muscle strength, walking ability, and exercise endurance improved. Although relatively rare, clinicians should consider the possibility of a thromboembolic event in KTWS patients. Integrated rehabilitation can help such patients to recover function. CONCLUSION In conclusion, although rare, patients with KTWS may experience central nervous system vascular malformations and accompanying stroke. It is necessary to investigate whether such patients have any neurological or comorbid abnormalities. Even in the subacute or chronic period after neurological insult, integrated rehabilitation programs can lead to structural and functional enhancement.
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