Functional Loss of the Tumor Suppressor Gene RASAL1 Through Formation of Aberrant Splice Variant RASAL1-004 as a Novel Oncogenic Mechanism in Thyroid Cancer

Background: RASAL1 is a prominent tumor suppressor gene through inactivating RAS, whose functional loss has been widely found to play an important role in thyroid cancer and occurs usually through its genetic and epigenetic inactivation. In this study, we intended to explore aberrant alternative splicing (AS) as an important novel mechanism for the oncogenic inactivation of RASAL1 in thyroid cancer. Methods: We used comprehensive bioinformatic and molecular experimental approaches to identify and characterize aberrant alternative splice variants of RASAL1 . This included structural and functional investigation of the potentially oncogenic splice variants of RASAL1, with a focus on exploring the molecular mechanisms and clinical impacts on thyroid cancer. Results: We identified an aberrant alternative splice variant of RASAL1 , known as RASAL1-004 , that commonly compromised the function of RASAL1 in thyroid cancer. Specifically, we found common skipping of exon 18.1 in RASAL1 , leading to the abundant formation of transcript RASAL1-004 in cancer, which was significantly associated with poor survival of patients with thyroid cancer. Mechanistically, Argonaute2 regulates exon 18.1 splicing by binding to the response element in exon 17 containing CCAGCC motif, promoting RASAL1-004 formation. The exon 18.1 skipping caused a conformational change in the RNA structure of RASAL1-004 at the junction of exons 17 and 18, resulting in ribosome stalling, halting RASAL1 translation. This reduced RAS GTPase-activating-like protein 1 (RASAL1 protein) synthesis, consequently leading to the functional loss of RASAL1 . Compared with RASAL1-001 , the canonical wild-type RASAL1 transcript, the absence of exon 18.1 in RASAL1-004 also conformationally altered the pleckstrin homology domain of RASAL1 protein, which, as we demonstrated, led to the loss of the ability of RASAL1 to localize with cell membrane, thereby impairing its RAS-inactivating function. We further demonstrated that compared with RASAL1-001 , RASAL1-004 displayed impaired RAS-signaling pathway-suppressing and cancer cell-suppressing functions. Conclusions: This study identified a novel RASAL1 -impairing mechanism, alternative to the classically known genetic and epigenetic mechanisms, for the inactivation of the tumor suppressor gene RASAL1 through aberrant AS to form RASAL1-004 with impaired protein translation. This represents a new oncogenic mechanism in thyroid cancer, with novel cancer biological, prognostic, and therapeutic-targeting implications in thyroid cancer.