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Long noncoding RNAs (lncRNAs) that map to intragenic regions of the human genome with the same (intronic lncRNAs) or opposite orientation (antisense lncRNAs) relative to protein-coding mRNAs have been largely dismissed from biochemical and functional characterization due to the belief that they are mRNA precursors, byproducts of RNA splicing or simply transcriptional noise. In this work, we used a custom microarray to investigate aspects of the biogenesis, processing, stability, evolutionary conservation, and cellular localization of ∼ 6,000 intronic lncRNAs and ∼ 10,000 antisense lncRNAs. Most intronic (2,903 of 3,427, 85%) and antisense lncRNAs (4,945 of 5,214, 95%) expressed in HeLa cells showed evidence of 5' cap modification, compatible with their transcription by RNAP II. Antisense lncRNAs (median t1/2 = 3.9 h) were significantly (p < 0.0001) more stable than mRNAs (median t1/2 = 3.2 h), whereas intronic lncRNAs (median t1/2 = 2.1 h) comprised a more heterogeneous class that included both stable (t1/2 > 3 h) and unstable (t1/2 < 1 h) transcripts. Intragenic lncRNAs display evidence of evolutionary conservation, have little/no coding potential and were ubiquitously detected in the cytoplasm. Notably, a fraction of the intronic and antisense lncRNAs (13 and 15%, respectively) were expressed from loci at which the corresponding host mRNA was not detected. The abundances of a subset of intronic/antisense lncRNAs were correlated (r ≥ |0.8|) with those of genes encoding proteins involved in cell division and DNA replication. Taken together, the findings of this study contribute novel biochemical and genomic information regarding intronic and antisense lncRNAs, supporting the notion that these classes include independently transcribed RNAs with potentials for exerting regulatory functions in the cell.

Changing the abundance of transcripts by regulated RNA degradation is a critical step in the control of various biological pathways. Recently, genome-wide inhibitor-free technologies for determining RNA stabilities in mammalian cells have been developed. In these methods, endogenous RNAs are pulse labeled by uridine analogs [e.g., 4-thiouridine (4sU), 5-etyniluridine (EU) and 5'-bromo-uridine (BrU)], followed by purification of labeled de novo RNAs. These technologies have revealed that the specific half-life of each mRNA is closely related to its physiological function. Genes with short-lived mRNAs are significantly enriched among regulatory genes, while genes with long-lived mRNAs are enriched among housekeeping genes. This review describes the recent progress of experimental procedures for measuring RNA stability.

Argonaute proteins are key players in microRNA (miRNA) processing and function. Next to their role as RISC effector proteins mediating target silencing, they actively participate in miRNA biogenesis and increase miRNA abundance by a yet unidentified mechanism. It has been hypothesized that this increase in steady-state miRNA levels might be due to a stabilizing effect of Argonaute proteins, but this has not been analyzed so far due to a lack of test systems. Here, we provide two approaches to estimate miRNA stability and factors affecting it: in cells lacking Ago2, endogenous miRNA guide strand degradation and half-lives can be assessed using Actinomycin D or α-Amanitin. In turn, miRNA passenger strands can serve as a model in wildtype cells to ascertain the impact of miRNA stability factors. We provide evidence that Argonaute proteins stabilize mature miRNAs in a slicing-independent manner. Transcriptional inhibition reveals reduced half-lives of multiple endogenous miRNA guide strands in cells lacking Ago2. This effect is reversible upon the reconstitution of Argonaute expression. Correspondingly, overexpression of Argonaute proteins decelerates miRNA degradation and increases miRNA half-life. Taken together, this study employs two model systems to identify factors altering miRNA stability and provides evidence how Argonaute proteins post-transcriptionally elevate mature miRNA levels via increasing miRNA stability.

Purpose: To isolate and to characterize cells expressing RNA polymerase II tagged with green fluorescent protein for analyses of the effects of ionizing radiation on transcription in living cells., Materials and Methods: We introduced an alpha-amanitin-resistant mutation into a vector encoding the largest subunit of RNA polymerase II tagged with green fluorescent protein (GFP-pol). Cell lines stably expressing functional GFP-pol were isolated under selection with alpha-amanitin from a Chinese hamster cell line, CHO-K1, and a radiation-sensitive mutant CHO cell line, XR-1., Results: We tested the functionality of the fusion protein in vivo by determining RNA synthesis activity by incorporation of nucleoside analogues. Both CHO-K1 and XR-1 cells expressing GFP-pol had properties similar to those of their respective parental cell lines, indicating that GFP-pol is functional., Conclusions: These stable lines might prove useful for analyses of the roles of transcription after ionizing radiation.