Fig. 3: Characterization of the endonucleolytic RNA fragments.

a Xrn1 digestion of total RNA extracts from dom34 mutant cells in the presence or absence of polynucleotide kinase (PNK) in vitro. 8% PAGE followed by northern blot analysis using probe prA. The 5S rRNA served as a loading control and 5.8S rRNA as a positive control of Xrn1 treatment. b Flow chart illustrating the method used for 3′-RACE as described in ref. ³¹ with minor modifications according to McGlincy and Ingolia³⁰ (see the Methods section). c PCR products obtained from 3′-RACE and separated on a 2% agarose gel. Purified DNAs for sequencing are indicated by an arrowhead. Prior to PCR, cDNAs were produced from total RNA from ski2, ski2/dom34 mutant cells expressing mRNA1. Control is made of total RNA from ski2/dom34 mutant cells without mRNA1 expression. d Sequences obtained after 3′-RACE performed on ski2 and ski2/dom34 total RNA. 100% of sequenced clones (omitting a residual 5S rRNA-linker amplification detected) have this DNA sequence. 5′-NGD DNA sequence (in green) and linker sequence (in magenta). Below, the site of mRNA1 is shown before and after the cleavage producing the 3’-NGD RNA B4 and the 3′-extremity of the 5′-NGD RNA confirmed by 3′-RACE. Source data are provided as a Source Data file.