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

<b><i>Background:</i></b> <i>RASAL1</i> 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 <i>RASAL1</i> in thyroid cancer. <b><i>Methods:</i></b> We used comprehensive bioinformatic and molecular experimental approaches to identify and characterize aberrant alternative splice variants of <i>RASAL1</i>. This included structural and functional investigation of the potentially oncogenic splice variants of <i>RASAL1,</i> with a focus on exploring the molecular mechanisms and clinical impacts on thyroid cancer. <b><i>Results:</i></b> We identified an aberrant alternative splice variant of <i>RASAL1</i>, known as <i>RASAL1-004</i>, that commonly compromised the function of <i>RASAL1</i> in thyroid cancer. Specifically, we found common skipping of exon 18.1 in <i>RASAL1</i>, leading to the abundant formation of transcript <i>RASAL1-004</i> 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 <i>RASAL1-004</i> formation. The exon 18.1 skipping caused a conformational change in the RNA structure of <i>RASAL1-004</i> at the junction of exons 17 and 18, resulting in ribosome stalling, halting <i>RASAL1</i> translation. This reduced RAS GTPase-activating-like protein 1 (RASAL1 protein) synthesis, consequently leading to the functional loss of <i>RASAL1</i>. Compared with <i>RASAL1-001</i>, the canonical wild-type <i>RASAL1</i> transcript, the absence of exon 18.1 in <i>RASAL1-004</i> 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 <i>RASAL1-001</i>, <i>RASAL1-004</i> displayed impaired RAS-signaling pathway-suppressing and cancer cell-suppressing functions. <b><i>Conclusions:</i></b> This study identified a novel <i>RASAL1</i>-impairing mechanism, alternative to the classically known genetic and epigenetic mechanisms, for the inactivation of the tumor suppressor gene <i>RASAL1</i> through aberrant AS to form <i>RASAL1-004</i> 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.