U3 small nuclear RNA can be psoralen-cross-linked in vivo to the 5' external transcribed spacer of pre-ribosomal-RNA.
U3 small nuclear RNA is hydrogen-bonded to high molecular weight nucleolar RNA and can be isolated from greater than 60S pre-ribosomal ribonucleoprotein particles, suggesting that it is involved in processing of ribosomal RNA precursors (pre-rRNA) or in ribosome biogenesis. Here we have used in vivo psoralen cross-linking to identify the region of pre-rRNA interacting with U3 RNA. Quantitative hybridization selection/depletion experiments with clones of rRNA-encoding DNA (rDNA) and cross-linked nuclear RNA showed that all of the cross-linked U3 RNA was associated with a region that includes the external transcribed spacer (ETS) at the 5' end of the human rRNA precursor. To further identify the site of interaction within the approximately 3.7-kilobase ETS, Southern blots of rDNA clones were sandwich-hybridized with cross-linked RNA and then probed for cross-linked U3 RNA. These experiments showed that U3 RNA was cross-linked to a 258-base sequence between nucleotides +438 and +695, just downstream of the ETS early cleavage site (+414). The localization of U3 to this region of the rRNA precursor was not expected from previous models for a base-paired U3-rRNA interaction and suggests that U3 plays a role in the initial pre-rRNA processing event.
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City 66103.
R L
RL
Maser
J P
JP
Calvet
D015139:Blotting, Southern; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D004275:DNA, Ribosomal / Q000235:genetics / Q000378:metabolism*; D011564:Furocoumarins / Q000494:pharmacology*; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D005810:Multigene Family; D009693:Nucleic Acid Hybridization; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D012342:RNA, Small Nuclear / Q000378:metabolism*; D014158:Transcription, Genetic
D003432:Cross-Linking Reagents; D004275:DNA, Ribosomal; D011564:Furocoumarins; D012322:RNA Precursors; D012342:RNA, Small Nuclear