Experimental validation of the regulated expression of large numbers of non-coding RNAs from the mouse genome

Abstract

Recent large-scale analyses of mainly full-length cDNA libraries generated from a variety of mouse tissues indicated that almost half of all representative cloned sequences did not contain an apparent protein-coding sequence, and were putatively derived from non-protein-coding RNA (ncRNA) genes. However, many of these clones were singletons and the majority were unspliced, raising the possibility that they may be derived from genomic DNA or unprocessed pre-mRNA contamination during library construction, or alternatively represent nonspecific "transcriptional noise." Here we show, using reverse transcriptase-dependent PCR, microarray, and Northern blot analyses, that many of these clones were derived from genuine transcripts of unknown function whose expression appears to be regulated. The ncRNA transcripts have larger exons and fewer introns than protein-coding transcripts. Analysis of the genomic landscape around these sequences indicates that some cDNA clones were produced not from terminal poly(A) tracts but internal priming sites within longer transcripts, only a minority of which is encompassed by known genes. A significant proportion of these transcripts exhibit tissue-specific expression patterns, as well as dynamic changes in their expression in macrophages following lipopolysaccharide stimulation. Taken together, the data provide strong support for the conclusion that ncRNAs are an important, regulated component of the mammalian transcriptome.

Figure 1.

(A) Distribution of exon number in mRNAs and putative ncRNAs. (B) Distribution of the number of As within the following 20 nt of the adjacent 3′ genomic sequence. The black bars are mRNAs (protein coding and flanking exonic sequences), and the white bars are putative ncRNAs.

Figure 2.

Tissue profiling of putative ncRNAs. RNA was isolated from 20 mainly adult tissues, and corresponding cDNA probes were prepared and hybridized to RIKEN 20K-2 microarrays as described in Methods. (A) Relative frequency histogram of signal intensity of mRNAs and filtered ncRNAs in the microarrays. Mean signal intensity values for each sequence were calculated for individual tissues relative to negative control cDNAs from Arabidopsis (Accession nos. X98108, X13611, X90769, Z99707, AF004393, Z49777, Q03943, U58284) and a positive control (β-actin). To compare the distribution of signals, the fluorescence values for mRNAs and ncRNAs were grouped into bins of every 100 relative fluorescence units (RFU). The number of signals within each bin was then converted to a percentage of the total number of signals across all tissues for each category. To distinguish real from background levels, the background frequency for each bin (as estimated by the Arabidopsis controls) was subtracted from each of the remaining three groups, and the resulting relative frequency values were plotted. (B) Tissue distribution of up-regulated ncRNAs. Here 178 differentially expressed ncRNAs with >2-fold up-regulation relative to the day 17.5 whole embryo reference tissue were identified using Welch ANOVA with Bonferroni multiple testing correction (P = 0.01). To determine tissue distribution, the number of up-regulated ncRNAs within each tissue was calculated and plotted. (C) Hierarchical clustering of up-regulated ncRNAs. Here 178 differentially expressed ncRNAs showing greater than twofold up-regulation in expression were hierarchically clustered according to tissue expression using the Cluster tool (Eisen et al. 1998). Groups of clones showing tissue-specific expression patterns are indicated: (1) testis; (2) muscle; (3) central nervous system; (4) thymus; (5) pancreas; (6) liver; (7) enteric tract. Tissues labeled with “n” are neonatal tissues. The primary data used for the cluster analysis, including the GenBank ID numbers and the relative intensity ratios of the differentially expressed ncRNA sequences, are given in Supplemental Table 5.

Figure 3.

Northern blot analysis of the size and tissue distribution of selected ncRNAs. Details of the methods can be found in the Supplemental material. The full-length cDNA probes of the eight ncRNAs used in this analysis were obtained from the DNABook (Hayashizaki and Kawai 2004). The corresponding GenBank IDs (and sizes) are: (A) AK035433 (1318 bp); (B) AK028310 (2905 bp); (C) AK017092 (765 bp); (D) AK040014 (139 bp); (E) AK040058 (1141 bp); (F) AK014924 (1799 bp). Total RNA was used, and blots were exposed in a PhosphorImager cassette for 24 h at 4°C. The positions and sizes (in kilobases) of molecular mass markers are shown.

Figure 4.

Dynamics of the expression of putative ncRNAs during macrophage activation by LPS. Panels 1-4 show quantitative real-time PCR profiles for 13 selected ncRNAs, as follows: (1) AK008526 (- ♦ -), AK017043 (—▪—), and AK007024 (- ▴ - -); (2) AK019555 (- - ▴ - -), AV079268 (—♦—) and AK017039 (- ▪ - -); (3) AK009126 (- ▪ -), AK033985 (—♦—), AK017432 (x - -), and AK007998 (- - ▴ - -); (4) AK008218 (- ▴ -), AK018521 (—♦—), and AK035433 (- - ▪ -). Panels 5-8 show examples of expression patterns of selected sense-antisense pairs of ncRNAs with their cognate protein-coding transcripts in macrophages. The sense-antisense pairs are mRNAs (- - ♦ - -) and ncRNAs (—▪—), as follows: (5) Cacnb4/AK035433 (correlation coefficient = -0.77, P-value = 0.23); (6) Ubxd6 (Rep-8)/AK017432 (correlation coefficient = -0.83, P-value = 0.17); (7) Atp5l/AK017092 (correlation coefficient = -0.79, P-value = 0.21); (8) Slc22a6/AK018521 (correlation coefficient = 0.54, P-value = 0.46). RNA was isolated at time 0, and at 2, 7, and 24 h after LPS stimulation. The abscissa indicates the average fold change in ncRNA/mRNA expression compared to time 0.