Many uridine modifications have been tested with zero to minimal decrease in RNase 4 activity, including pseudo-, N1-methyl-pseudo-, 5-methoxy, and dihydrouridine species (Ψ, m1Ψ, mo5U, and D). To efficiently digest RNAs containing these modifications, especially m1Ψ, we recommend increasing the added volume from 1 µl to 3 µl of RNase 4 (50,000 U/ml) in equivalent 10 µg RNA digests.  Since RNase 4 endonucleolytic activity requires the 2´-hydroxyl of the uridine nucleotide, modifications such as 2´ -O methyluridine (Um) completely inhibit RNase 4 activity.