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U6 snRNA function in nuclear pre-mRNA splicing: A phosphorothioate interference analysis of the U6 phosphate backbone
Abstract
U6 snRNA is essential for and may participate in the catalysis of pre-mRNA splicing. Extensive mutational analyses in several systems have identified nucleotides essential for U6 function in splicing; however, relatively little is known regarding the role of the U6 phosphate backbone. We previously described a mutation in a nematode U6 snRNA that causes it to be used as a splicing substrate within the spliceosome. This unusual reaction has made it possible to apply modification interference analysis to U6 function. Here, we have used phosphorothioate substitution to identify pro-R oxygens throughout the U6 backbone that are necessary for the first and/or second catalytic steps of splicing. Four pro-R oxygens are important for the first step; of these only two appear to be required. One additional pro-R oxygen is uniquely required for the second step. The two pro-R oxygens critical for the first step of splicing are in the helix 1b U2/U6 interaction region and the intramolecular stem-loop of U6, respectively. A comparison of the positions of these two pro-R oxygens with those found to be critical for autocatalytic excision of a group II intron suggests a possible functional similarity between U6 snRNA and domain V of group II introns.
... In breast, ovarian, and endometrial cancers, GPER1 has been linked to proliferation, metastasis, and poor prognosis [30]. GPER1 is also suspected to trigger resistance to endocrine therapy in breast cancer, since tamoxifen has been shown to act as a GPER1 agonist [31,32]. However, while a majority of these studies have proposed a pro-tumorigenic action of GPER1, others have reported antiproliferative activities [30]. ...
MicroRNAs play critical roles through their impact on posttranscriptional gene regulation. In cancer, they can act as oncogenes or tumor suppressors and can also function as biomarkers. Here, we describe a method for robust characterization of estrogen-regulated microRNA profiles. The activity of estrogen is mediated by two nuclear receptors, estrogen receptor alpha and estrogen receptor beta, and a transmembrane G-protein coupled estrogen receptor 1. This chapter details how to prepare cells for optimal estrogen response, directions for estrogen treatment, RNA extraction, different microRNA profiling approaches, and subsequent confirmations.
... The equivalents in the spliceosome are found in the U6/U6atac snRNAs [37,42,43]. The evolutionary conserved elements are the ACAGAGA box corresponding to J2/3 and an AGC triad in a bulged internal stem loop that corresponds to domain DV. ...
In eukaryotes, RNA trans-splicing is a significant RNA modification process for the end-to-end ligation of exons from separately transcribed primary transcripts to generate mature mRNA. So far, three different categories of RNA trans-splicing have been found in organisms within a diverse range. Here, we review trans-splicing of discontinuous group II introns, which occurs in chloroplasts and mitochondria of lower eukaryotes and plants. We discuss the origin of intronic sequences and the evolutionary relationship between chloroplast ribonucleoprotein complexes and the nuclear spliceosome. Finally, we focus on the ribonucleoprotein supercomplex involved in trans-splicing of chloroplast group II introns from the green alga Chlamydomonas reinhardtii. This complex has been well characterized genetically and biochemically, resulting in a detailed picture of the chloroplast ribonucleoprotein supercomplex. This information contributes substantially to our understanding of the function of RNA-processing machineries and might provide a blueprint for other splicing complexes involved in trans- as well as cis-splicing of organellar intron RNAs.
... The U6 snRNA ACAGAGA sequence of Helix I is involved in 5′ splice site selection, and in promoting both steps of the splicing reaction (Luukkonen and Seraphin 1998;Mefford and Staley 2009), and the U2 snRNA sequence opposing this ACAGAGA segment pairs with a region of the intron to position the branch site residue. Catalytically essential metal ion-binding sites have been identified at the catalytic AGC triad at the base of the U6 ISL, a bulged uridine in the ISL, and nucleotides at the 3ʹ end of the ACAGAGA loop (Gordon et al. 2000;Yu et al. 1995;Yuan et al. 2007;Yean et al. 2000;Sontheimer et al. 1997). ...
Intron removal during splicing of precursor pre-mRNA requires assembly of spliceosomal small nuclear (sn)RNAs into catalytically competent conformations to promote two transesterification reactions. U2 and U6 snRNA are the only snRNAs directly implicated in pre-mRNA splicing catalysis, but rearrangement and remodeling steps prior to catalysis require numerous proteins. Previous studies have shown that the protein-free U2-U6 snRNA complex adopts two conformations characterized by four and three helices surrounding a central junction in equilibrium. To analyze the role of the central junction in positioning the two helices critical for formation of the active site, we used ensemble time-resolved fluorescence resonance energy transfer to measure distances between fluorophores at selected locations in constructs representing the protein-free human U2-U6 snRNA complex. Data describing four angles in the four-helix conformer suggest the complex adopts a tetrahedral geometry; addition of Mg2+ results in shortening of the distances between neighboring helices, indicating compaction of the complex around the junction. In contrast, the three-helix conformer shows a closer approach between the two helices bearing critical elements, but addition of Mg2+ widens the distance between these stems. Presence of Mg2+ also enhances the steady state fraction of the three-helix conformer found to be active in spliceosomes. Although the central junction assumes a significant role in orienting helices, in neither conformer, with or without Mg2+, are the critical helices positioned sufficiently close to favor interaction, implying that a major role of spliceosomal remodeling proteins is to overcome such distances to create and stabilize a catalytically active fold.
... This model stated, by analogy to protein phosphoryl transfer mechanisms, that two divalent metal ions situated 3.9 Å apart stabilize the nucleophile and the leaving group during transesterification. Two groups used phosphorothioate substitutions to identify functionally important nonbridging oxygen atoms in the U6 phosphoribose backbone (Fabrizio and Abelson 1992;Yu et al. 1995), and subsequent thiophilic metal ion rescue experiments revealed that the U6 ISL coordinates the two Mg 2+ ions required for catalysis via five nonbridging phosphate oxygens (Yean et al. 2000;Fica et al. 2013). Electron density in the active site of recent spliceosome cryo-EM structures has been modeled as metal ions (Wan et al. 2016a;Yan et al. 2016Yan et al. , 2017Fica et al. 2017;Zhang et al. 2017). ...
Removal of introns from precursor messenger RNA (pre-mRNA) and some non-coding transcripts is an essential step in eukaryotic gene expression. In the nucleus, this process of RNA splicing is carried out by the spliceosome, a multi-megaDalton macromolecular machine whose core components are conserved from yeast to humans. In addition to many proteins, the spliceosome contains five uridine-rich small nuclear RNAs (snRNAs) that undergo an elaborate series of conformational changes to correctly recognize the splice sites and catalyze intron removal. Decades of biochemical and genetic data, along with recent cryo-EM structures, unequivocally demonstrate that U6 snRNA forms much of the catalytic core of the spliceosome and is highly dynamic, interacting with three snRNAs, the pre-mRNA substrate, and >25 protein partners throughout the splicing cycle. This review summarizes the current state of knowledge on how U6 snRNA is synthesized, modified, incorporated into snRNPs and spliceosomes, recycled, and degraded.
... 52,64,65 Specifically, the internal bulge provides catalytically important ligands for two metals (M1 and M2) that are directly involved in both splicing steps ( Figure 3C,D) and also interacts with a conserved U2-U6 helical region (termed helix IB) to from a structurally conserved catalytic triplex ( Figure 3E,F). 21,59,63,66 The dual functions of this internal bulge of U6 suggest that this region of the spliceosome contains both ancient structures and conserved dynamic motions that are essential for properly orienting the substrate and activating catalysis of the pre-mRNA substrate. 56,67 ...
Over billions of years of evolution, nature has embraced proteins as the major workhorse molecules of the cell. However, nearly every aspect of metabolism is dependent upon how structured RNAs interact with proteins, ligands, and other nucleic acids. Key processes, including telomere maintenance, RNA processing, and protein synthesis, require large RNAs that assemble into elaborate three-dimensional shapes. These RNAs can i) act as flexible scaffolds for protein subunits, ii) participate directly in substrate recognition, and iii) serve as catalytic components. Here, we juxtapose the near atomic level interactions of three ribonucleoprotein (RNP) complexes: ribonuclease P (involved in 5’- pre-tRNA processing), the spliceosome (responsible for pre-mRNA splicing), and telomerase (an RNA-directed DNA polymerase that extends the ends of chromosomes). The focus of this perspective is to profile structural and dynamic roles of RNAs at the core of these enzymes, highlighting how large RNAs contribute to molecular recognition and catalysis.
... Two transcripts encoding U6 snRNA, a non-coding small nuclear RNA, were repressed (Supplementary Table S4). U6 snRNA is critical for pre-mRNA splicing 35 . ...
In eukaryotic cells, RNA polymerase III is highly conserved and transcribes housekeeping genes such as ribosomal 5S rRNA, tRNA and other small RNAs. The RPC5-like subunit is one of the 17 subunits forming RNAPIII and its exact functional roles in the transcription are poorly understood. In this work, we report that virus-induced gene silencing of transcripts encoding a putative RPC5-like subunit of the RNA Polymerase III in a model species Nicotiana benthamiana had pleiotropic effects, including but not limited to severe dwarfing appearance, chlorosis, nearly complete reduction of internodes and abnormal leaf shape. Using transcriptomic analysis, we identified genes and pathways affected by RPC5 silencing and thus presumably related to the cellular roles of the subunit as well as to the downstream cascade of reactions in response to partial loss of RNA Polymerase III function. Our results suggest that silencing of the RPC5L in N. benthamiana disrupted not only functions commonly associated with the core RNA Polymerase III transcripts, but also more diverse cellular processes, including responses to stress. We believe this is the first demonstration that activity of the RPC5 subunit is critical for proper functionality of RNA Polymerase III and normal plant development.
The cleavage and ligation reactions at RNA phosphodiester bonds are the central reactions catalyzed by enzymes in critical cellular regulatory pathways. In pre-mRNA splicing, two phospho-transesterifications result in the right mRNA for protein synthesis with the intervening intron removed as a lariat structure. The lariat RNA is then debranched by an enzyme that specifically acts on this 2′-5′-branched RNA. Following debranching, some of these introns that include pre-microRNA sequences can be processed by Dicer that cleaves the RNA to provide microRNAs. Dicer and Drosha, enzymes that act on much bigger primary transcripts, are both RNase III-like enzymes that cleave the RNA phosphodiester linkage. All these reactions are in related pathways, and the RNA phosphodiester bonds are most likely cleaved with the aid of two metal ions, yet the active sites that host these could be composed entirely of RNA or entirely of protein, or possibly a hybrid of the two. Where unknown, it is possible to estimate some of these active site architectures through homology to closely related enzymes. Better insight into these related process and active sites will play a key role in leveraging these important RNA regulatory processes for molecular medicine.
Splicing of group II introns is essential for the metabolism of many organisms (Michel et al. 1989; Michel and Ferat 1995). These ubiquitous introns play a critical role in the processing of mitochondrial genes from plants, fungi, and yeast. Group II introns and RNA molecules resembling them are abundant in euglena and other lower eukaryotes, and they have even been identified in prokaryotes. It has been proposed that, through reactions analogous to the reverse of splicing, excised introns can migrate and introduce themselves into new genomes that may not even ordinarily contain introns (Lambowitz and Belfort 1993; Schmidt et al. 1994). Thus, in addition to their function in RNA splicing, group II introns have the capability for involvement in other biochemical transformations. The apparent complexity of their structure and active-site chemistry has fueled interest in the mechanism of group II intron catalysis. This chapter attempts to describe recent work on group II intron chemistry and its foundation in structural features of the folded RNA.
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