RNA-binding protein SAM68 interacts with endocytic proteins and actin cytoskeleton modulators

Pankivskyi, SV
Senchenko, NV
Busko, PB
Rynditch, AV
Dopov. Nac. akad. nauk Ukr. 2020, 5:103-109
Section: Biology
Language: English

SAM68 is a nuclear RNA-binding protein involved in the regulation of mRNA processing. SAM68 overexpression is observed in multiple types of cancer. Recently, the possible link between RNA-binding protein SAM68 and scaffold protein ITSN1 has been identified. The aim of the study was to confirm the probability of direct binding bet ween SAM68 and ITSN, analyze the effect of ITSN1 on SAM68-mediated alternative splicing, and identify novel SAM68 partners among endocytic proteins and actin cytoskeleton modulators. The interactions were revealed in pull-down assays using purified recombinant proteins or cell lysates. ITSN1 knockdown in HeLa cells was performed using the shRNA approach. The expression of isoforms produced by alternative splicing was measured using RT-PCR. It was demonstrated that SAM68 directly interacted with ITSN1 in vitro. Next, it was found that ITSN1 knockdown in HeLa cells induced SRSF1 intron 3 retention increasing the expression of the proto-oncogenic isoform of SRSF1 by three times. It was also shown that SH3 domains of AMPH1, BIN1, CTTN1, TKS4, and TKS5 precipitated SAM68 from lysate of 293 cells. As a result, SAM68 directly binds to ITSN1 and interacts with endocytic proteins and actin cytoskeleton modulators, whereas SAM68-mediated splicing in HeLa cells may be regulated by ITSN1.

Keywords: alternative splicing, ITSN1, SAM68, SRSF1

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