Characterization of stable RNAs from the resected intestinal tissues of individuals with either Crohn's disease or ulcerative colitis

Abstract

Circular RNAs reminiscent of viroids and the human hepatitis delta virus have been proposed as possible nonconventional pathogens responsible for Crohn's disease and ulcerative colitis, two inflammatory bowel diseases. Consequently, RNA was extracted from various areas of intestinal tissues from individuals with either Crohn's disease or ulcerative colitis as well as several appropriate control diseases, and analyzed by two-dimensional gel electrophoresis. No circular viroid-like RNAs (< 1500 nucleotides) were detected, confirming a previous report that was limited to the investigation of small RNAs (< 300 nucleotides). However, three small, unusually stable, linear RNAs were shown to be associated to both Crohn's disease and ulcerative colitis tissues: a specific 28S ribosomal RNA cleavage product characterized previously; a 5.8S ribosomal RNA conformer; and a fragment homologous to transcripts from DNA CpG islands. The two last RNAs were detected prior to visible morphological tissue alterations, suggesting that they are produced early during the inflammation and that they have value as molecular diagnostic tools for the inflammatory bowel diseases. The potential cellular mechanisms producing these RNAs and their involvement in inflammatory bowel disease are discussed.

Fig. 1

2D-PAGE fractionation of RNA extracted from tissues using the acidified phenol procedure. (A) Analysis of a mixture of both circular (338 nucleotides) and linear RNA peach latent mosaic viroids. C, circular transcripts; 1, 2, 3, 4, and 5, linear transcripts corresponding to 745, 621, 462, 338, and 283 nucleotides, respectively. In each case 1 mu;g of RNA was applied to the gel. (B) RNA extracted from the intestinal tissue of an individual affected by Crohn’s disease. On this panel, several species of RNA are identified. (C) RNA extracted from the intestinal tissue of an individual with a diverticulitis. This gel was overstained to ensure detection of any small amounts of off-diagonal migrating species. For Figs. 1B and 1C, 200 mu;g of RNA was applied to the gel. The directions of migration under the native (1st) and the denaturing (2nd) conditions are shown in Fig. 1C.

Fig. 2

Electron microscopy of (A) RNA Y and (B) RNA Z under denaturing conditions. The arrows indicate RNA molecules.

Fig. 3

Enzymatic analysis of RNA Y and Z by 5′ end labelling. RNA were phosphorylated directly (lanes 1 and 3) or after a prior dephosphorylation (lanes 2 and 4); RNA Y (lanes 1 and 2) and RNA Z (lanes 3 and 4). Adjacent to the gel are the positions of RNA standards (length in nucleotides). ori, origin of migration; XC, position of xylene cyanol.