Pre-mRNA processing enhancer (PPE) elements from intronless genes play additional roles in mRNA biogenesis than do ones from intron-containing genes

Most mRNA-encoding genes require introns for efficient expression in high eukaryotes. However, mRNAs can efficiently accumulate in the cytoplasm without intron excision if they contain cis-acting elements such as the post-transcriptional regulatory element (PRE) of hepatitis B virus (HBV), the constitutive transport element (CTE) of Mason–Pfizer monkey virus (MPMV), or the pre-mRNA processing enhancer (PPE) of herpes simplex virus' thymidine kinase (HSV-TK) gene. We compared the activities of these viral elements, the Rev-responsive element (RRE) of the human immunodeficiency virus (HIV), and the human c-Jun gene's enhancer (CJE), an element newly identified here, to enable expression of an intronless variant of the human β-globin gene. The PRE, PPE and CJE from naturally intronless genes, but not the CTE or RRE from intron-containing genes, significantly enhanced stability, 3′ end processing and cytoplasmic accumulation. When the transcripts included the β-globin gene's first intron, the PRE, PPE and CJE still enhanced mRNA biogenesis, in some cases without intron excision. Thus, elements enabling stability, 3′ end formation and nucleocytoplasmic export, not the presence of introns or their excision per se, are necessary for mRNA biogenesis. While the CTE and RRE primarily enhance nucleocytoplasmic export, PPE-like elements from naturally intronless genes facilitate polyadenylation as well

The Functions of Non-coding RNAs in rRNA Regulation

Ribosomes are ribonucleoprotein machines that decode the genetic information embedded in mRNAs into polypeptides. Ribosome biogenesis is tightly coordinated and controlled from the transcription of pre-rRNAs to the assembly of ribosomes. Defects or disorders in rRNA production result in a number of human ribosomopathy diseases. During the processes of rRNA synthesis, non-coding RNAs, especially snoRNAs, play important roles in pre-rRNA transcription, processing, and maturation. Recent research has started to reveal that other long and short non-coding RNAs, including risiRNA, LoNA, and SLERT (among others), are also involved in pre-rRNA transcription and rRNA production. Here, we summarize the current understanding of the mechanisms of non-coding RNA-mediated rRNA generation and regulation and their biological roles

The USTC co-opts an ancient machinery to drive piRNA transcription in C. elegans.

Piwi-interacting RNAs (piRNAs) engage Piwi proteins to suppress transposons and nonself nucleic acids and maintain genome integrity and are essential for fertility in a variety of organisms. In Caenorhabditis elegans, most piRNA precursors are transcribed from two genomic clusters that contain thousands of individual piRNA transcription units. While a few genes have been shown to be required for piRNA biogenesis, the mechanism of piRNA transcription remains elusive. Here we used functional proteomics approaches to identify an upstream sequence transcription complex (USTC) that is essential for piRNA biogenesis. The USTC contains piRNA silencing-defective 1 (PRDE-1), SNPC-4, twenty-one-U fouled-up 4 (TOFU-4), and TOFU-5. The USTC forms unique piRNA foci in germline nuclei and coats the piRNA cluster genomic loci. USTC factors associate with the Ruby motif just upstream of type I piRNA genes. USTC factors are also mutually dependent for binding to the piRNA clusters and forming the piRNA foci. Interestingly, USTC components bind differentially to piRNAs in the clusters and other noncoding RNA genes. These results reveal the USTC as a striking example of the repurposing of a general transcription factor complex to aid in genome defense against transposons

A Pre-mRNA–Associating Factor Links Endogenous siRNAs to Chromatin Regulation

In plants and fungi, small RNAs silence gene expression in the nucleus by establishing repressive chromatin states. The role of endogenous small RNAs in metazoan nuclei is largely unknown. Here we show that endogenous small interfering RNAs (endo-siRNAs) direct Histone H3 Lysine 9 methylation (H3K9me) in Caenorhabditis elegans. In addition, we report the identification and characterization of nuclear RNAi defective (nrde)-1 and nrde-4. Endo-siRNA–driven H3K9me requires the nuclear RNAi pathway including the Argonaute (Ago) NRDE-3, the conserved nuclear RNAi factor NRDE-2, as well as NRDE-1 and NRDE-4. Small RNAs direct NRDE-1 to associate with the pre-mRNA and chromatin of genes, which have been targeted by RNAi. NRDE-3 and NRDE-2 are required for the association of NRDE-1 with pre-mRNA and chromatin. NRDE-4 is required for NRDE-1/chromatin association, but not NRDE-1/pre-mRNA association. These data establish that NRDE-1 is a novel pre-mRNA and chromatin-associating factor that links small RNAs to H3K9 methylation. In addition, these results demonstrate that endo-siRNAs direct chromatin modifications via the Nrde pathway in C. elegans

Binding of hnRNP L to the Pre-mRNA Processing Enhancer of the Herpes Simplex Virus Thymidine Kinase Gene Enhances both Polyadenylation and Nucleocytoplasmic Export of Intronless mRNAs

Liu and Mertz (Genes Dev. 9:1766-1780, 1995) previously identified a 119-nt pre-mRNA processing enhancer (PPE) element within the herpes simplex virus type 1 thymidine kinase gene that enables intron-independent gene expression in higher eukaryotes by binding heterogeneous nuclear ribonucleoprotein L (hnRNP L). Here, we identify a 49-nt subelement within this PPE that enhanced stability, polyadenylation, and cytoplasmic accumulation of transcripts synthesized in CV-1 cells from an intronless variant of the human β-globin gene when present in two or more tandem copies. This 2×TK49 PPE also enhanced (i) the efficiency of polyadenylation of intronless β-globin RNA in a cell-free polyadenylation system and (ii) the kinetics of nucleocytoplasmic export of an intronless variant of adenovirus major late leader region RNA in Xenopus oocytes. This 2×TK49 PPE bound only hnRNP L. Analysis of 2×TK49 PPE mutants showed a strong positive correlation existed between binding hnRNP L and enhancement of intronless β-globin gene expression. hnRNP L was found to associate with both the mRNA export factor TAP and the exon-exon junction complex protein Aly/REF. Thus, we conclude that hnRNP L plays roles in enhancing stability, polyadenylation, and nucleocytoplasmic export; it does so, at least in part, by directly recruiting to intronless PPE-containing RNAs cofactors normally recruited to intron-containing RNAs

The human gene contains elements that enhance stabilization, 3′ end processing and cytoplasmic accumulation of intronless transcripts

<p><b>Copyright information:</b></p><p>Taken from "Pre-mRNA processing enhancer (PPE) elements from intronless genes play additional roles in mRNA biogenesis than do ones from intron-containing genes"</p><p>Nucleic Acids Research 2005;33(7):2215-2226.</p><p>Published online 20 Apr 2005</p><p>PMCID:PMC1083424.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () Autoradiogram of quantitative S1 nuclease mapping analysis of β-globin-like RNAs accumulated in the nucleus (N) and cytoplasm (C) 48 h after co-transfection of CV-1PD cells with the indicated plasmids and pRSV-Tori. The S1 nuclease mapping probes were the 5′ end-labeled ones shown in . The samples were analyzed as described in the legend to . The numbers below the lanes are mean ± SEM values of data obtained from three independent experiments similar to the one shown here. () Autoradiogram of quantitative S1 nuclease mapping analysis of the 3′ ends of the β-globin-like RNAs accumulated in the nucleus and cytoplasm of CV-1PD cells. Portions of the RNA samples from the experiment shown in (A) were mapped with the 3′ end-labeled probe shown in . The numbers below the lanes are mean ± SEM values of data obtained from three independent experiments similar to the one shown here; they were calculated as described in the legend to . () The β-globin-like RNAs accumulated in the cytoplasm are unspliced. A portion of the RNA sample from the experiment shown in (A), lane 6, was reverse-transcribed and amplified by PCR as described in . Shown here is a photograph of an ethidium bromide-stained 1% agarose gel in which the PCR products were electrophoresed. The rightmost lane contains a 1 kilobase pair DNA ladder. () Autoradiogram of quantitative S1 nuclease mapping analysis showing that a 201 nt region of is sufficient to enhance cytoplasmic accumulation of intronless β-globin-like transcripts. COS-M6 cells were transfected with the indicated plasmids and processed as described in (A)

Effects of the presence of a PRE, PPE, CTE or RRE in obviating the intron requirement for efficient 3′ end processing and cytoplasmic accumulation of human β-globin-like RNA

<p><b>Copyright information:</b></p><p>Taken from "Pre-mRNA processing enhancer (PPE) elements from intronless genes play additional roles in mRNA biogenesis than do ones from intron-containing genes"</p><p>Nucleic Acids Research 2005;33(7):2215-2226.</p><p>Published online 20 Apr 2005</p><p>PMCID:PMC1083424.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () Autoradiogram of quantitative S1 nuclease mapping analysis of the human β-globin-like RNAs accumulated in the nucleus and cytoplasm of CV-1PD cells transfected with the plasmids shown in . CV-1PD cells were co-transfected with 2 μg of the indicated plasmid along with 1 μg of the SV40 T antigen-encoding plasmid pRSV-Tori. Nuclear (N) and cytoplasmic (C) RNAs were harvested 48 h later and analyzed by concurrent quantitative S1 nuclease mapping with the 5′ end-labeled β-globin and β-actin probes shown in . Transfection and RNA fractionation efficiencies were analyzed as described in Methods. The amount of β-globin-like RNA accumulated in the cytoplasm was internally normalized to the amount of cellular β-actin RNA present in the same sample. The numbers shown below the lanes are the percentages relative to the amount of β-globin-like RNA accumulated in the cytoplasm of cells transfected in parallel with β-β1(+)2(+) RNA, with normalization as well to the relative amounts of replicated β-globin plasmid DNA present in the nuclear fractions of those samples. These numbers are mean ± SEM values of data obtained from five independent experiments similar to the one shown here. () Autoradiogram of quantitative S1 nuclease mapping analysis of the 3′ ends of the β-globin-like RNAs accumulated in the nucleus and cytoplasm of CV-1PD cells. The RNA samples from the experiment in panel A were analyzed with the 3′ end-labeled probe shown in . The numbers below the pairs of lanes are mean ± SEM values of the cleaved RNA (N + C) divided by the cleaved plus uncleaved RNA (N + C) times 100% of data obtained from three independent experiments similar to the one shown here. () The β-globin-like RNAs accumulated in the cytoplasm are unspliced. Portions of the cytoplasmic RNA samples from the experiment shown in (A) were reverse-transcribed and then amplified by PCR with the primers described in Methods (+RT, lanes 3, 6, 9, 12 and 15). As controls, PCR amplification reactions were performed on the RNA samples without prior reverse transcription (−RT, lanes 2, 5, 8, 11 and 14) and on the plasmid DNAs used in the transfections (DNA, lanes 4, 7, 10, 13 and 16). Shown here is a photograph of an ethidium bromide-stained 1% agarose gel in which the PCR products were electrophoresed. Lane 1 contained 1 kilobase pair ladder DNA as size markers