Polyadenylylated nuclear RNA contains branches

Abstract

A highly charged component can be isolated from a total RNase T2 digest of nuclear polyadenylylated RNA from HeLa cells that is separable from caps by (dihydroxyboryl)aminoethyl-cellulose chromatography. Chemical and enzymatic analyses show that the component contains a 2'-5' phosphodiester bond that creates a branch at the 2'-hydroxyl group of one nucleotide already linked to an adjoining nucleotide through the usual 3'-5' phosphodiester bond.p_5'y3'_p N3-P-5'X2'/ This structure was confirmed by analysis of a similar component isolated from nuclease P1 digests of the same nuclear polyadenylylated RNA. Branches occur in roughly 10% of nuclear polyadenylylated RNAs, including those >1OS in size, but are absent from cytoplasmic polyadenylylated RNA. Possible implications for branches as intermediates in mRNA processing are discussed.The processing ofnuclear transcripts into mature mRNA in eukaryotic cells involves a variety of covalent modifications that include polyadenylylation, capping, methylation, and splicing of RNAs. The transient existence of the intermediates in these cellular processes and the low concentrations of individual mRNA precursors has greatly restricted their isolation in quantities sufficient for chemical characterization.The polyadenylylated heterogeneous nuclear RNA (hnRNA) of animal cells represents a precursor population containing many different mRNA sequences. It is sufficiently long lived to provide a source of labeled RNAs that undergo similar covalent modifications and can be examined for processing intermediates free of artifacts associated with the use of inhibitors.During an investigation of the "cap" structures in RNase digests of polyadenylylated nuclear RNA, we detected another highly negatively charged component. Chemical and enzymatic analyses indicate that it is derived from a RNA molecule containing a 2'-5' phosphodiester bond at the 2'-OH group of a nucleotide already joined by the normal 3'-5' phosphodiester bond to an adjacent nucleotide. The absence of such structures from polyadenylylated cytoplasmic mRNA raises the possibility that branched RNA may arise during RNA processing in the cell nucleus. We describe here the isolation and characterization of the structure at which RNA branching occurs. METHODSApproximately 8 x 107 cells in late logarithmic phase were labeled with 50 mCi of 32P04 (1 Ci = 37 GBq) at a concentration of4 x 106 cells per ml for 4 hr as described (1). Cell fractionation was by the method of Penman (2), except that the DNase step was omitted and the supernatant from the detergent wash ofthe nuclei was discarded. The cytoplasmic fraction was freed of mitochondria by centrifugation for 10 min at 10,000 x g. Nuclear and cytoplasmic RNA was extracted by the hot phenol/ NaDodSO4 method (3).Poly(A)+RNA was obtained by three cycles of binding to oligo(dT)-cellulose (P-L Biochemicals, type 7) as described (4). The 60°C heating step was included for each binding to minimize contamination with nonpolyadenylylated RNA. The efficiency of the third bind...

Precise localization of m6A in Rous sarcoma virus RNA reveals clustering of methylation sites: implications for RNA processing.

“…Upon cleavage at a 5' splice site, the 5' end of the intron forms a 5'-+2' bond with an internal nucleotide in the intron, thus creating a branch at that nucleotide. Similar branched structures have been characterized in the nuclear polyadenylated RNA of HeLa cells (37). In more than 80% of these structures, the base at which branching occurs is adenosine, frequently within either GA*C or AA*C sequences, where * designates the branched residue.…”

Alternate trans splicing in Trypanosoma equiperdum: implications for splice site selection.

“…The minRNA can be removed from this structure with (HeLa cell nucleus) debranching enzyme. Polyadenylated nuclear RNA (in yeast and higher eucaryotic cells) has been shown to contain branched RNA intermediates which are formed during cis splicing (21,24,25,34).…”

An excised SV40 intron accumulates and is stable in Xenopus laevis oocytes.

“…However, cellular RNAs from higher eukaryotes corresponding in size and sequence to introns removed from p-globin (Zeitlin and Efstratiadis 1984) and immunoglobulin (Coleclough and Wood 1984) premRNAs have been described. The existence of branched RNA structures was first described in nuclear RNA by Wallace and Edmonds (1983), who postulated that they may be RNA-processing intermediates. Identification of yeast (Domdey et al 1984;Rodriguez et al 1984), pglobin (Zeitlin and Efstratiadis 1984), and SV40 large tumor antigen (Noble et al 1987) introns from intact cells existing in circular or lariat form was subsequently reported.…”