Plant small RNAs are subject to various modifications. Previous reports revealed widespread 3′ modifications (truncations and non-Templated tailing) of plant miRNAs when the 2′-O-methyltransferase HEN1 is absent. However, non-Templated nucleotides in plant heterochromatic siRNAs have not been deeply studied, especially in wild-Type plants. We systematically studied non-Templated nucleotide patterns in plant small RNAs by analyzing small RNA sequencing libraries from Arabidopsis, tomato, Medicago, rice, maize and Physcomitrella. Elevated rates of non-Templated nucleotides were observed at the 3′ ends of both miRNAs and endogenous siRNAs from wild-Type specimens of all species. 'Off-sized' small RNAs, such as 25 and 23 nt siRNAs arising from loci dominated by 24 nt siRNAs, often had very high rates of 3′-non-Templated nucleotides. The same pattern was observed in all species that we studied. Further analysis of 24 nt siRNA clusters in Arabidopsis revealed distinct patterns of 3′-non-Templated nucleotides of 23 nt siRNAs arising from heterochromatic siRNA loci. This pattern of non-Templated 3′ nucleotides on 23 nt siRNAs is not affected by loss of known small RNA 3′-end modifying enzymes, and may result from modifications added to longer heterochromatic siRNA precursors.
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