3′-terminal region of the nascent MALAT1 contains evolutionarily conserved sequences that are predicted to interact before 3′-end maturation. (A) mascRNA is located immediately downstream from the MALAT1 A-rich tract (boxed in purple). A 7-nt long mascRNA 3′ trailer (boxed in black), which is highly conserved among vertebrates, is fully complementary to the A-rich tract. (B) Schematic of the intronless β-globin reporter construct containing a cytomegalovirus promoter, a human βΔ1,2 gene, and a bovine growth hormone poly(A) site (BGH pA). In ENE-containing reporters, the ENE (green), the A-rich tract (purple), and the mascRNA (black) are inserted upstream of the poly(A) site. The RNase P cleavage site is marked by an arrowhead. (C) Schematic of the MALAT1 ENE + A-rich tract shows the triple helical structure formed at the 3′ end of mature MALAT1. The U-rich internal loops are in green and the A-rich tract is in purple. (D) The human mascRNA is predicted to fold into a tRNA-like structure with an extended acceptor stem that is formed through base-pairing interactions between the upstream A-rich tract and the downstream mascRNA 3′ trailer. A single A residue is predicted to bulge immediately upstream of the RNase P cleavage site. Nucleotides targeted for mutagenesis are outlined in red boxes. In C and D, nucleotide 8343 of the human MALAT1 is indicated. (E) Northern blots probed for β-globin and control neomycin resistance (NeoR) transcripts (top) were quantitated by normalizing the β-globin signals to those of NeoR (bottom). Reporter accumulation with the wild-type MALAT1 ENE + A-rich tract + mascRNA was set at 1. Relative accumulation was the average of at least three independent experiments ± SD.
