New small nuclear RNA gene-like transcriptional units as sources of regulatory transcripts

Abstract

By means of a computer search for upstream promoter elements (distal sequence element and proximal sequence element) typical of small nuclear RNA genes, we have identified in the human genome a number of previously unrecognized, putative transcription units whose predicted products are novel noncoding RNAs with homology to protein-coding genes. By elucidating the function of one of them, we provide evidence for the existence of a sense/antisense-based gene-regulation network where part of the polymerase III transcriptome could control its polymerase II counterpart.

Figure 1. CENP-F as 21A-Specific Molecular Target

(A) Human CENP-F gene structure as resulting from GI:89161185 (region 212843155 - 212904537). (B) The positions of the 21A antisense homologous regions are reported together with their percentage of identity. (C) Sequence alignment of 21A/CENP-F homologous regions.

Figure 2. Pol III-Dependent Synthesis of Novel Transcription Units

(A) Northern blot analysis of human skin fibroblasts and HeLa cells. Results show two bands: the first (detected at about 300 nucleotides) being the 21A endogenous product and the second (of a very high molecular mass) representing CENP-F mRNA. (B) 21A-specific RT-PCR amplification. As expected for nonpolyadenylated transcripts, an efficient amplification product was obtained only in the random hexamers-primed reactions. (C) Promoter activity transfection assay. A specific luciferase-silencing hairpin is transcribed by six PSE/DSE-dependent promoter elements (11A [H1 RNA], 14A, 21A, 29A, 38A, 51A). A view of the silencing constructs including the hairpin nucleotide sequence is enclosed. The promoter region encompasses the putative pol III type 3 regulatory regions (TATA box, PSE, and DSE). pGL3 + pRL, negative control; pSHAG-U6, canonical pol III promoter; No promoter, hairpin without PSE/DSE-dependent promoter thus resulting transcriptionally inactive. (D and E) Promoter-activity transfection assay in presence/absence of 20 μM ML-60218 cell-permeable pol III inhibitor or 10 μg/ml α-amanitin pol II–specific inhibitor. Results are reported as luciferase emission of treated versus untreated samples. (F) Real-time RT-PCR analysis of transcript levels for 21A and 29A transcription units, two known pol III–transcribed genes (7SK and 5S rRNA), and one pol II–transcribed gene (c-Myc) in ML-60218 treated/untreated cells, as resulting after normalization of each treated sample with its untreated counterpart. All the measures were referred to a ML-60218 unaffected pol II housekeeping gene (GAPDH). (G) Real-time RT-PCR analysis of the RNA level of two known pol II–transcribed (c-Myc and GAPDH) and one pol III–transcribed (7SK) genes in α-amanitin treated/untreated cells as resulting after normalization of each treated sample with its untreated counterpart. All the measures were referred to an α-amanitin unaffected pol III gene (5S rRNA).

Figure 3. 21A-Driven CENP-F Expression Regulation

(A–D) 21A constructs. d, DSE Element; p, PSE Element; p21A, whole transcription unit; p21A-1, upstream, DSE-containing region; p21A-2, transcription region; pMock, empty vector; t, TATA box. (E–H) CENP-F protein expression level after 0, 24, 48, and 72 h of constructs transfection. Full triangle, anti-CENP-F antibody; Full circle, anti-tubulin antibody (for protein loading normalization); Striped columns, quantitative determination of CENP-F expression modulation as determined by Western blot analysis; Full columns, quantitative determination of CENP-F mRNA expression modulation as determined real-time RT-PCR analysis. (I–N) 21A RNA level in transfected samples indicating that the exogenous 21A expression inversely correlates with CENP-F protein expression. (O) Dissociation curve of 21A amplification products. A, 21A-transfected HeLa cells; B, untransfected HeLa cells showing the very low basal 21A transcription level.

Figure 4. Modification of Cell Proliferation Rate in 21A-Overexpressing HeLa Cells

(A) Proliferation inhibition of HeLa cells after 48 h of 21A constructs transfection. Results emphasize the specificity of the Alu Jb-containing region as a source of proliferation inhibitory transcripts. (B) Proliferation increase of HeLa cells after 48 h of pAnti-21A and si21A transfection. siEx-FABP, unrelated chicken-specific siRNA (negative control). (C and D) Constructs structures. Anti-21A, the transcript region is inverted and the construct maintains 21A promoter as well as its termination site. si21A, siRNA 21A-specific. (E and F) CENP-F protein expression level after 0, 24, and 48 h of constructs transfection. Full triangle, anti-CENP-F antibody; full circle, anti-tubulin antibody; striped columns, quantitative determination of CENP-F expression modulation as determined by Western blot analysis; full columns, quantitative determination of CENP-F mRNA expression modulation as determined real-time RT-PCR analysis. (G and H) 21A RNA level in transfected samples indicating that the endogenous 21A expression is inhibited after 24 h of anti-21A/si21A transfection.

Figure 5. Mouse NIH-3T3 Cell Proliferation Rate after Transfection of 21A Constructs

No proliferation decrease was observed.

Figure 6. Real-Time RT-PCR Analysis of 21A Endogenous RNA in Different Cell Types with Respect to the Less Abundant Samples (293T for 21A RNA and Fibroblasts for 5S rRNA)

Striped columns, 21A RNA; full columns, 5S rRNA. The dissociation curve of 21A amplification product in PBL is reported in the inset.