Abstract
<title>Abstract</title> <p> <bold>Background</bold> Alternative transcript isoforms contribute to functional diversification of the proteome by generating protein variants with distinct structural and regulatory properties. Dicer1e is a naturally occurring truncated protein isoform of human Dicer (hDicer) detected in both physiological and cancer-associated contexts; however, its biochemical activity and biological function remain poorly understood. <bold>Methods</bold> Dicer1e was ectopically expressed in HEK293T cells and hDicer-deficient HEK293T NoDice cells. Its biochemical properties were characterized using <italic>in vitro</italic> RNA cleavage assays, while its structural features were predicted using AlphaFold3. Cellular effects were evaluated by RNA sequencing, quantitative proteomics, RT-qPCR, immunoblotting, and proliferation assays. <bold>Results</bold> Dicer1e retained Mg²⁺-dependent RNase activity but did not generate canonical ~ 21–22-nucleotide hDicer cleavage products, instead producing heterogeneous RNA fragments, consistent with altered substrate positioning following loss of the Platform and PAZ domains. Structural modeling revealed preservation of the catalytic RNase III core together with a unique, evolutionarily conserved 13-amino-acid N-terminal extension, which is predicted to contribute to the distinct structural properties of the isoform. Dicer1e expression induced extensive, context-dependent remodeling of cellular regulatory networks. Although transcriptomic and proteomic datasets exhibited limited overlap at the level of individual genes and proteins, both analyses converged at the biological pathway level, highlighting alterations in RNA metabolism, intracellular organization, vesicle-mediated transport, cellular signaling, and proliferation. These molecular responses differed substantially between HEK293T and HEK293T NoDice cells, indicating that endogenous hDicer modulated the cellular consequences of Dicer1e expression. Consistent with these observations, Dicer1e promoted proliferation more prominently in hDicer-deficient cells than in cells expressing endogenous hDicer. <bold>Conclusions</bold> These findings identify Dicer1e as a specialized RNase with biochemical and regulatory properties distinct from those of full-length hDicer. More broadly, this study demonstrates that alternative transcript isoforms can generate functionally specialized protein variants with biological properties distinct from those of their canonical counterparts, highlighting the importance of considering alternative <italic>DICER1</italic> transcripts as potential contributors to RNA regulation. </p>