In our study, we analyzed HSPA5 binding motifs peak calling and noted that HSPA5 regulated most genes via 5′UTR alternative splicing and introns and in a manner dependent on AG-rich sequences, where the HSPA5-AGAG interaction might play an important role in regulating alternative splicing of NAFLD-related genes. This evidence concerns the gene HSPA5 and metabolic dysfunction-associated steatotic liver disease.