Ancestral RNA: The RNA biology of the eukaryotic ancestor
Our knowledge of RNA biology within eukaryotes has exploded over the last five years. Within new research we see that some features that were once thought to be part of multicellular life have now been identified in several protist lineages. Hence, it is timely to ask which features of eukaryote RNA biology are ancestral to all eukaryotes. We focus on RNA-based regulation and epigenetic mechanisms that use small regulatory ncRNAs and long ncRNAs, to highlight some of the many questions surrounding eukaryotic ncRNA evolution.
The U3 snoRNA is an exceptional box C/D snoRNA, which is involved in pre-rRNA processing without directing chemical modifications. We report here on a comprehensive computational survey resulting in U3 sequences for more than 90 additional eukaryotes. This extended data basis is used to improve the secondary structure models. The detailed investigation of the structural variation of U3 snoRNAs turns out to be much more extensive than previously thought. Many fungal U3 genes, in addition, contain introns. U3 promoters are snRNA-like but show substantial variations even between related species.
The signal recognition particle (SRP) is a ribonucleoprotein complex which participates in the targeting of protein to cellular membranes. The RNA component of the SRP has been found in all domains of life, but the size of the molecule and the number of RNA secondary structure elements vary considerably between the different phylogenetic groups. We continued our efforts to identify new SRP RNAs, compare their sequences, discover new secondary structure elements, conserved motifs, and other properties. We found additional proof for the variability in the apical loop of helix 8, and we identified several bacteria which lack all of their SRP components. Based on the distribution of SRP RNA features within the taxonomy, we suggest seven alignment groups: Bacteria with a small (4.5S) SRP RNA, Bacteria with a large (6S) SRP RNA, Archaea, Fungi (Ascomycota), Metazoa group, Protozoa group, and Plants. The proposed divisions improve the prediction of more distantly related SRP RNAs and provide a more inclusive representation of the SRP RNA family. Updates of the Rfam SRP RNA sequence collection are expected to benefit from the suggested groupings.
Sculpting AMPA receptor formation and function by alternative RNA processing
AMPA receptors are ion channel tetramers that mediate fast excitatory neurotransmission in vertebrate brains. AMPAR functional properties as well as receptor biogenesis in the endoplasmic reticulum (ER) is influenced by RNA processing events, including adenosine-to-inosine RNA editing and alternative splicing. Recoded sites line interfaces of subunit polypeptides, and are therefore ideally positioned to modulate receptor assembly. Moreover, the genomic arrangement of the R/G editing site within the splice donor of the alternative flip/flop exons may facilitate a cross-talk between these elements. Regulated mRNA recoding in response to neuronal activity would have the scope to sculpt AMPAR tetramers and in turn shape the response properties of a neuron.
An up-close look at the pre-mRNA 3’-end processing complex
Polyadenylation is a nearly universal mRNA processing step in eukaryotic gene expression and it takes place in a macromolecular machinery termed the mRNA 3’ processing complex. In a recent study, we reported the purification and comprehensive characterization of functional mammalian mRNA 3’ processing complexes. Our study defined the protein composition of this machinery, revealed new potential links between mRNA 3’ processing and other cellular processes, and characterized basic structural features of the “core” mRNA 3’ processing complex. These results provided new insights into the mechanism of mRNA 3’-end formation.
Nuclear export of mRNA and its regulation by ubiquitylation
The export of mRNA from nucleus to cytoplasm is a key regulatory step in the expression of RNA polymerase II genes in eukaryotes, and thus, a variety of human diseases are manifested by abnormal mRNA export. Therefore, a large number of studies over many years have been directed towards elucidating the regulatory mechanisms of mRNA export. These studies have identified several mRNA export-associated factors and delineated their regulatory networks. Intriguingly, mRNA export has also been shown to be regulated by ubiquitylation, a post-translational modification that targets proteins for degradation or transport and has been linked to different cellular processes such as cell cycle progression, DNA repair, transcription and intracellular trafficking. While the mechanisms of actions of ubiquitylation in different cellular processes are relatively well-established, it is not clearly understood how mRNA export is regulated by ubiquitylation. Here, we highlight the recent advances of mRNA export and its regulation by ubiquitylation or the enzymes controlling this post-translational modification.
How to control miRNA maturation? Co-activators and co-repressors take the stage
In this point of view we discuss the role of co-activators and co-repressors of maturation of miRNA precursors, the possibility that their functions are post-translationally regulated by different signaling pathways, and their potential role in the miRNA-dependent control of cell proliferation and differentiation.
Assaying microRNA loss-of-function phenotypes in mammalian cells: Emerging tools and their potential therapeutic utility
MicroRNAs are small, non-coding RNAs that are increasingly appreciated to play critical roles in the modulation of gene expression. In mammalian cells, our knowledge regarding the full impact of microRNAs on cellular behavior remains fragmentary. This has been due, in significant part, to the limited availability of experimental tools for studying microRNA loss-of-function phenotypes. Recently, several strategies for achieving this goal have been developed. Here, we discuss these methodologies for inhibiting specific microRNAs in mammalian cells both in vitro and in vivo, compare and contrast the strengths and weaknesses of these approaches, and speculate regarding the future impact of these antagonists on the treatment of human diseases such as cancer. These emerging techniques enable the attenuation of microRNA function in a manner that is quite sequence-specific, relatively long-lasting, and increasingly cost-effective. As such, some of these advances hold great promise in terms of their eventual utility as therapeutic agents.
The epithelial splicing factors ESRP1 and ESRP2 positively and negatively regulate diverse types of alternative splicing events
Cell-type and tissue-specific alternative splicing events are regulated by combinatorial control involving both abundant RNA binding proteins as well as those with more discrete expression and specialized functions. Epithelial Splicing Regulatory Proteins 1 and 2 (ESRP1 and ESRP2) are recently discovered epithelial-specific RNA binding proteins that promote splicing of the epithelial variant of the FGFR2, ENAH, CD44, and CTNND1 transcripts. To cataloge a larger set of splicing events under the regulation of the ESRPs we profiled splicing changes induced by RNA interference-mediated knockdown of ESRP1 and ESRP2 expression in a human epithelial cell line using the splicing sensitive Affymetrix Exon ST1.0 Arrays. Analysis of the microarray data resulted in the identification of over a hundred candidate ESRP regulated splicing events. We were able to independently validate 37 of these targets by RT-PCR. The ESRP regulated events encompass all known types of alternative splicing events, most prominent being alternative cassette exons and splicing events leading to alternative 3’ terminal exons. Importantly, a number of these regulated splicing events occur in gene transcripts that encode proteins with well-described roles in the regulation of actin cytoskeleton organization, cell-cell adhesion, cell polarity, and cell migration. In sum, this work reveals a novel list of transcripts differentially spliced in epithelial and mesenchymal cells, implying that coordinated alternative splicing plays a critical role in determination of cell type identity. These results further establish ESRP1 and ESRP2 as global regulators of an epithelial splicing regulatory network.
Interaction of yeast eIF4G with spliceosome components: Implications in pre-mRNA processing events
As evidenced from mammalian cells the eukaryotic translation initiation factor eIF4G has a putative role in nuclear RNA metabolism. Here we investigate whether this role is conserved in the yeast Saccharomyces cerevisiae. Using a combination of in vitro and in vivo methods, we show that, similar to mammalian eIF4G, yeast eIF4G homologues, Tif4631p and Tif4632p, are present both in the nucleus and the cytoplasm. We show that both eIF4G proteins interact efficiently in vitro with UsnRNP components of the splicing machinery. More specifically, Tif4631p and Tif4632p interact efficiently with U1 snRNA in vitro. In addition, Tif4631p and Tif4632p associate with protein components of the splicing machinery, namely Snu71p and Prp11p. To further delineate these interactions, we map the regions of Tif4631p and Tif4632p that are important for the interaction with Prp11p and Snu71p and we show that addition of these regions to splicing reactions in vitro has a dominant inhibitory effect. The observed interactions implicate eIF4G in aspects of pre-mRNA processing. In support of this hypothesis, deletion of one of the eIF4G isoforms results in accumulation of un-spliced precursors for a number of endogenous genes, in vivo. In conclusion these observations are suggestive of the involvement of yeast eIF4G in pre-mRNA metabolism.
MicroRNA-125a represses cell growth by targeting HuR in breast cancer
MicroRNAs (miRNAs) are a class of naturally occurring, small, non-coding RNAs that control gene expression during development, normal cell function, and disease. Although there is emerging evidence that some miRNAs can function as oncogenes or tumor suppressors, there is limited understanding of the role of miRNAs in cancer. In this study, we observed that the expression of miR-125a was inversely correlated with HuR expression in several different breast carcinoma cell lines. HuR is a stress-induced RNA binding protein whose expression is elevated or localization perturbed in several different cancers. Increased cytoplasmic localization of HuR is a prognostic marker in breast cancer. Real time PCR and gene reporter assays indicated that HuR was translationally repressed by miR-125a. Re-establishing miR-125a expression in breast cancer cells decreased HuR protein level and inhibited cell growth. Using MCF-7 breast cancer cells, we further clarified that miR-125a inhibited cell growth via a dramatic suppression of cell proliferation and promotion of apoptosis. In addition, cell migration was also inhibited by miR-125a overexpression. Importantly, the repression of cell proliferation and migration engendered by miR-125a was partly rescued by HuR re-expression. Our results suggest that miR-125a may function as a tumor suppressor for breast cancer, with HuR as a direct and functional target.
The influence of Hfq and ribonucleases on the stability of the small non-coding RNA OxyS and its target rpoS in E. coli is growth phase dependent
OxyS is one of at least three small non-coding RNAs, which affect rpoS expression. It is induced under oxidative stress and reduces the levels of the stationary phase sigma factor RpoS. We analyzed the turn-over of OxyS and rpoS mRNA in early exponential and in stationary growth phase in different E. coli strains to learn more about the mechanisms of processing and about a possible impact of processing on growth-dependent regulation. We could not attribute a major role of RNase E, RNase III, PNPase or RNase II on OxyS turn-over in exponential growth phase. Only the simultaneous lack of RNase E, PNPase and RNase II activity resulted in some stabilization of OxyS in exponential growth phase, implying the action of multiple ribonucleases on OxyS turn-over. A major role of RNase E on OxyS stability was observed in stationary phase and was dependent on the presence of the RNA binding protein Hfq and of DsrA, one of the other small RNAs binding to rpoS mRNA. Our data also confirm a role of RNase III in rpoS turn-over, however, only in exponential growth phase. We conclude that OxyS and rpoS mRNA processing is influenced by different RNases and additional factors like Hfq and DsrA and that the impact of these factors is strongly dependent on growth phase.