SmY RNAs are a family of ~70–90 nt small nuclear RNAs found in nematodes. In C. elegans, SmY RNAs copurify in a small ribonucleoprotein (snRNP) complex related to the SL1 and SL2 snRNPs that are involved in nematode mRNA trans-splicing. Here we describe a comprehensive computational analysis of SmY RNA homologs found in the currently available genome sequences. We identify homologs in all sequenced nematode genomes in class Chromadorea. We are unable to identify homologs in a more distantly related nematode species, Trichinella spiralis (class: Dorylaimia), and in representatives of non-nematode phyla that use trans-splicing. Using comparative RNA sequence analysis, we infer a conserved consensus SmY RNA secondary structure consisting of two stems ﬂanking a consensus Sm protein binding site. A representative seed alignment of the SmY RNA family, annotated with the inferred consensus secondary structure, has been deposited with the Rfam RNA families database.
Deep in the heart of Ohio, scientists from across the Midwest gathered in October to share their latest findings and highlight the strength of RNA research in the heartland. Represented were researchers from Delaware, Indiana, Kentucky, Michigan, Ohio, Pennsylvania and West Virginia. With over 220 participants, the 2008 annual Rustbelt RNA Meeting (RRM) was the largest gathering of this group in its 10-year history. The success of this year’s RRM lies on the extraordinary efforts of organizers Dawn Chandler (Ohio State University) and Girish Shukla (Cleveland State University)
Messenger RNA export from the nucleus to the cytoplasm plays an essential role in linking transcription to translation and consequently regulation of protein expression. mRNA export requires a series of events: pre-mRNA processing, ribonucleoprotein targeting to the NPC (nuclear pore complexes), and translocation through nuclear pores to the cytoplasm. Interestingly, the conventional nuclear export machinery, exportins and the Ran GTPase, is not required for mRNA export. Instead, a protein complex consisting of a number of RNA binding proteins is essential for this event including the Aly/REF protein. Phosphoinositide signaling regulates a variety of cellular functions including pre-mRNA splicing and mRNA export. In fact, a phospholipase C-dependent inositol polyphosphate kinase pathway is required for efficient mRNA export. Recently, we showed that Aly is a physiological target of nuclear phosphoinositide-3-kinase (PI3K) signaling, which regulates Aly localization as well as Aly function in cell proliferation and mRNA export through nuclear Akt-mediated phosphorylation and phosphoinositide association. Hence, water-soluble inositol polyphosphates and phosphatidylinositol lipids play pivotal roles in modulating mRNA export.
The current understanding of Ded1p/DDX3 homologs from yeast to human
DExD/H-box RNA helicases are involved in almost all steps of the eukaryotic mRNA biogenesis. The DEAD-box protein Ded1p/DDX3 is conserved from yeast to human. Various lines of genetic and biochemical evidence have indicated a role of the yeast Ded1p in translation and, most likely, in precursor mRNA splicing as well. In contrast, although recent studies have begun to reveal the function of the mammalian DDX3 in translation control, its exact role remains vague and even controversial. Here, we review these findings and particularly discuss the functional aspects of Ded1p/DDX3 in translation control.
RNA-mediated chaperone type for de novo protein folding
Traditionally the principles of protein folding in vivo have been obtained largely from molecular chaperone studies. Through extensive studies on molecular chaperones, it becomes clear that most proteins can fold without their assistance in vivo, suggesting the existence of other chaperone types and mechanisms. Since all nascent polypeptides are linked to the ribosomes, protein folding in vivo should be understood in the context of vectorial protein synthesis and linkage of nascent chains to ribosome whose major components and basic structural frames are RNAs. Here we introduce a novel RNA-mediated chaperone type and a possible molecular basis for how RNAs can exert chaperoning effect on their linked aggregation-prone polypeptides. Extending potential chaperoning role of ribosome on the bound nascent polypeptide in a cis-acting manner, the findings further suggest a novel function of RNA molecules for protein folding inside cells. RNA interaction-mediated stabilization of folding intermediate against aggregation provides new insights into de novo protein folding in vivo and further extends the functional diversity of RNA molecules.
The biochemistry of RNA metabolism studied in situ
In vitro assays have contributed important insights into the mechanisms of RNA metabolism in cells. A growing collection of microscopy techniques is allowing the measurement of macromolecular binding and complex formation in the context of real cell. We will first discuss two of the more established techniques. Fluorescence resonance energy transfer (FRET) identifies binding partners, pairs of molecules residing in the same macromolecular complexes. The complimentary technique of fluorescence recovery after photobleaching (FRAP) measures the rates of binding and unbinding of those molecules in their complexes. A newer technique- in vitro FRAP- assesses the regulation of binding and complex formation by co-factors in the nucleus.
Translating organellar glutamine codons : A case by case scenario?
Aminoacyl-tRNAs are generally formed by direct attachment of an amino acid to tRNAs by aminoacyl-tRNA synthetases, but glutaminyl-tRNA (Q-tRNA) is an exception to this rule. Glutaminyl-tRNAGln (Q-tRNAQ) is formed by this direct pathway in the eukaryotic cytosol and in a small subset of bacteria, but is formed by an indirect transamidation pathway in most bacteria and archaea. To date it is almost impossible to predict what pathway generates organellar Q-tRNAQ in a given eukaryote. All eukaryotic genomes sequenced so far display a single glutaminyl-tRNA synthetase (QRS) gene which is at least responsible for the cytosolic QRS activity, as well as a gene coding for a mitochondrial ortholog of the essential GatB subunit of the tRNA-dependent amidotransferase (AdT). Indeed, QRS activity was found in protozoan mitochondria while AdT activity was characterized in plant organelles. The pathway for Q-tRNAQ synthesis in yeast and mammals mitochondria is still questionable.
The NFAR's (Nuclear Factors Associated with dsRNA): Evolutionarily conserved members of the dsRNA binding protein family
The dsRNA binding protein (DRBP) family comprise one or more evolutionarily conserved dsRNA-binding domains (DRBD) of approximately 65-68 amino acids, are found in both eukaryotes and prokaryotes and are even encoded by plants and viruses. DRBP’s do not recognize specific nucleotide sequences and primarily interact with approximately 11-16 base pairs present within A-form double helix RNAs, which can include ssRNA’s with extensive secondary structure. The DRBP family include TRBP (TAR RNA binding protein), PKR (protein kinase activated by dsRNA), PACT (Protein Activator of PKR), ADAR (Adenosine deaminases acting on RNA), and the RNase III family including DICER, which collectively play important roles in mRNA elongation, RNA interference (RNAi), mRNA editing, stability, splicing and/or export and translation. Here, we focus on the role of DRBP’s referred to as the NFARs (Nuclear Factors associated with dsRNA) which are translated from two major alternatively spliced products encoded from a single gene. Evidence indicates that the NFAR proteins play crucial roles in mRNA post-transcriptional regulation, including mRNA stability, export and translation and may also have an important function in host defense.
RNase P: increased versatility through protein complexity?
Ribonuclease P (RNase P) is an essential enzyme that catalyzes the 5’ endonucleolytic cleavage of precursor transfer RNAs (pre-tRNAs). It is found in all phylogenetic domains: bacteria, archaea, and eukaryotes. The bacterial enzyme consists of a single, catalytic RNA subunit and one small protein, while the archaeal and eukaryotic enzymes have 4-10 proteins in addition to a similar RNA subunit. The bacterial RNA acts as a ribozyme at high salt in vitro; however the added protein optimizes kinetics and makes specific contacts with the pre-tRNA substrate. The bacterial protein subunit also appears to be required for the processing of non-tRNA substrates by broadening recognition tolerance. In addition, the immense increase in protein content in the eukaryotic enzymes suggests substantially enlarged capacity for recognition of additional substrates. Recently intron-encoded box C/D snoRNAs were shown to be likely substrates for RNase P, and several lines of evidence suggest that the nuclear holoenzyme binds tightly to, and can cleave single-stranded RNA in a sequence dependent fashion. The possible involvement of RNase P in additional RNA processing or turnover pathways would be consistent with previous findings that RNase MRP, a variant of RNase P that has evolved to participate in ribosomal RNA processing, is also involved in turnover of specific messenger RNAs. The involvement of RNase P in multiple RNA processing pathways is discussed.
In human and mouse up to 72% of all genomic loci show evidence of transcription from both sense and antisense strands. The benefit of the resulting natural antisense transcripts (NATs) remains unclear, largely because of a lack of significant correlation between gene ontology and antisense transcription. Here we suggest that a well defined group of NATs may be identified based on structural characteristics. Specifically, these NATs are processed transcripts that are complementary to the corresponding processed sense transcripts in exonic regions. Recent reports have established that co-expressed sense transcripts/NATs are processed into short RNAs. These so called endo-siRNAs are found in both sense and antisense orientation and were hypothesized to mediate pseudogene silencing. Here we propose that NATs are biologically important sources of endo-siRNAs. We also propose that endo-siRNAs are essential components of a regulatory network to control the mutagenic burden that unfolds on nucleic acid level without direct consequences on protein expression.
Transcriptional control of microRNA expression in C. elegans: Promoting better understanding
Transcriptional regulation of microRNA (miRNA) expression is one of the least understood aspects of miRNA biogenesis. In C. elegans the list of miRNAs whose transcriptional control has been described in some detail is currently limited to four: let-7, lin-4, lsy-6, and mir-61. Each of these genes has been shown experimentally to be transcriptionaly regulated by cis- and/or trans-acting factors that either promote or inhibit expression. Additionally, computational methods based on conservation among miRNA genes have yielded predicted regulatory sequences in C. elegans that may function to regulate miRNA expression on a genome-wide scale.
Reconciling contradictory reports regarding translation of BACE1 mRNA: initiation mechanism is altered by different expression systems
We previously showed that translation from the rat BACE1 5' leader is cap-dependent and that four AUG codons (AUG1-4) in the 5' leader were bypassed, partially or completely, depending on the cell line. Two other groups reported comparable results with human BACE1 sequences in different cell lines, although different mechanisms were postulated. In contrast, a third group working with the human sequence reported that most translation events are initiated at AUG2. Using reporter constructs with the rat BACE1 5' leader in rat cells, we now show that this apparent discrepancy between studies can be explained by the use of different expression systems and differences in interpretation. When reporter constructs were transcribed in the nucleus, the upstream AUG codons did not affect translation, but when mRNAs were transcribed in the cytoplasm or when in vitro transcripts were transfected into cells, the upstream AUG codons inhibited translation. These findings suggest that when transcription occurs in the nucleus, the BACE1 mRNA initiates translation by a shunting mechanism. The results are less consistent with either leaky scanning or reinitiation and provide a caveat against the use of cytoplasmic expression systems or RNA transfection for analyses of translation initiation.
Y-box protein 1 (YB-1) is a multifunctional DNA/RNA-binding protein that regulates transcription and translation. The specificity of YB-1’s RNA binding and its consequences are unknown. Because expression and subcellular localization of YB-1 have been reported to be important in breast cancer, we determined the specificity and functional impact of YB-1 mRNA-binding in MCF7 breast cancer cells. We used YB-1 antibodies to immunoprecipitate YB-1 and microarray profiling to compare YB-1-bound and total poly(A) RNA. We demonstrated that YB-1 mRNA-binding was preferential. Transcript sequences significantly associated with this binding had high GC content. Selected YB-1 mRNA-binding targets were confirmed by QRT-PCR. However, downregulation of YB-1 levels by siRNA did not affect their RNA or protein expression. Thus, YB-1 has RNA-binding specificity; however, YB-1 binding does not necessarily regulate the stability or translation of its mRNA targets. Further study is needed to determine the functional consequences of selective YB-1 mRNA binding.
MicroRNAs identified in highly purified liver-derived mitochondria may play a role in apoptosis
MicroRNAs (miRNAs) are a class of small ~22 nt noncoding (nc) RNAs that regulate gene expression post-transcriptionally by direct binding to target sites on mRNAs. They comprise more than 1,000 novel species in mammalian cells and exert their function by modulating gene expression through several different mechanisms, including translational inhibition, and/or degradation of target mRNAs. Mitochondria maintain and express their own genome, which is distinct from the nuclear transcriptional and translational apparatus. Thus, they provide a potential site for miRNA mediated post-transcriptional regulation. To determine whether they maintain a unique miRNA population, we examined the miRNA profile from highly purified and RNase treated mitochondria from adult rat liver. Fifteen miRNAs were identified by microarray analysis of which, five were confirmed by TaqMan® 5’nuclease assays using rat specific probes. Functional analysis of the miRNAs indicated that they were not targeted to the mitochondrial genome nor were they complementary to nuclear RNAs encoding mitochondrial proteins. Rather, the mitochondria-associated miRNAs appear to be involved in the expression of genes associated with apoptosis, cell proliferation, and differentiation. Given the central role that mitochondria play in apoptosis, the results suggest that they may serve as reservoirs of select miRNAs that may modulate these processes in a coordinate fashion.
Known turnover and translation regulatory RNA-binding proteins interact with the 3’ UTR of SECIS-binding protein 2
The human selenoproteome is composed of ~25 selenoproteins, which cotranslationally incorporate selenocysteine, the 21st amino acid. Selenoprotein expression requires an unusual translation mechanism, as selenocysteine is encoded by the UGA stop codon. SECIS-binding protein 2 (SBP2) is an essential component of the selenocysteine insertion machinery. SBP2 is also the only factor known to differentiate among selenoprotein mRNAs, thereby modulating the relative expression of the individual selenoproteins. Here, we show that expression of SBP2 protein varies widely across tissues and cell types examined, despite previous observations of only modest variation in SBP2 mRNA levels. This discrepancy between SBP2 mRNA and protein levels implies translational regulation, which is often mediated via untranslated regions (UTRs) in regulated transcripts. We have identified multiple sequences in the SBP2 3’ UTR that are highly conserved. The proximal short conserved region is GU rich and was subsequently shown to be a binding site for CUG-BP1. The distal half of the 3’ UTR is largely conserved, and multiple proteins interact with this region. One of these proteins was identified as HuR. Both CUG-BP1 and HuR are members of the Turnover and Translation Regulatory RNA-Binding Protein family (TTR-RBP). Members of this protein family are linked by the common ability to rapidly effect gene expression through alterations in the stability and translatability of target mRNAs. The identification of CUG-BP1 and HuR as factors that bind to the SBP2 3’ UTR suggests that TTR-RBPs play a role in the regulation of SBP2, which then dictates the expression of the selenoproteome.
Structure and activity of the internal ribosome entry site within the human p27Kip1 5’-untranslated region
The cyclin dependent kinase inhibitor p27Kip1 is a key cell cycle regulatory protein that is often downregulated in cancer cells. The cellular levels of p27Kip1 are regulated, in part, through translational control mechanisms. The 5’-UTR of the p27Kip1 mRNA is known to harbor an IRES that may facilitate expression of p27Kip1 under conditions of stress such as loss of cell adhesion or growth factor and nutrient deprivation. The results presented here further characterize the p27Kip1 5’-UTR and its IRES activity. We confirm that the major transcription start site of the p27Kip1 gene produces an mRNA with a 5’-UTR of ~472 nucleotides. Other minor transcripts are also observed but the 472 nucleotide 5’-UTR displays the highest IRES activity. A structural model for the 472 nucleotide 5’-UTR was derived from nuclease digestion patterns coupled with MFOLD secondary structural prediction software. These results indicate that the 5’-UTR has significant secondary structure but also contains a large single-stranded loop that extends from nucleotides -31 to -66 relative to the start codon. Mapping of the ribosome entry window indicates that the ribosome is recruited to this single-stranded loop. The single-stranded loop also includes a U-rich sequence that has previously been shown to bind several proteins, including HuR. This is significant because HuR has previously been shown to inhibit p27Kip1 IRES activity and cause down regulation of endogenous p27Kip1 protein levels. Thus HuR may inhibit IRES activity by blocking the ribosome entry site.