Abstract
<title>Abstract</title> <p> <bold>Background:</bold> MicroRNAs (miRNAs) are key post-transcriptional regulators of gene expression that modulate diverse biological processes and are implicated in numerous diseases, including cancer. The terminal loop (TL) of precursor miRNAs (pre-miRNAs) is a key determinant of miRNA biogenesis, influencing both processing efficiency and interactions with RNA-binding proteins (RBPs). Despite its functional importance, the sequence architecture of human pre-miRNA TL regions has not been systematically characterized. <bold>Methods:</bold> TL region sequences from 955 human pre-miRNAs were analyzed for length distribution, nucleotide composition, positional nucleotide frequencies, enrichment of sequence motifs, and enrichment of experimentally defined RBP-binding motifs. <bold>Results:</bold> Human miRNA TL regions exhibited a distinct length distribution, peaking at 15 nucleotides. Uracil (U) was the most abundant nucleotide, followed by guanine (G), and nucleotide composition varied markedly across positions within TL regions, including relative enrichment of U and G at the 5′ end of TL regions. Motif enrichment analysis identified 24 enriched 3-mers, 61 enriched 4-mers, and 101 enriched 5-mers. The motifs CUG, CUGA, and UCUGA were the most significantly enriched motifs within the 3-mer, 4-mer, and 5-mer classes, respectively, with CUGA also showing significant enrichment at the 3′ end of TL regions. Several motifs matched previously reported RBP-binding motifs; however, none remained statistically significant after multiple-testing correction. In addition, several distinct human miRNAs belonging to the same miRNA clusters shared identical TL region sequences despite divergence in their precursor and mature miRNA sequences. <bold>Conclusions:</bold> Human pre-miRNA TL regions exhibit distinct sequence characteristics, including defined length distributions, positional nucleotide biases, and enriched sequence motifs. Together, these findings provide a comprehensive characterization of TL region sequence architecture across the human miRNome. </p>