gurken and the I factor retrotransposon RNAs share common localization signals and machinery

Drosophila gurken mRNA is localized by dynein-mediated transport to a crescent near the oocyte nucleus, thus targeting the TGFalpha signal and forming the primary embryonic axes. Here, we show that gurken and the I factor, a non-LTR retrotransposon, share a small consensus RNA stem loop of defined secondary structure, which forms a conserved signal for dynein-mediated RNA transport to the oocyte nucleus. Furthermore, gurken and the I factor compete in vivo for the same localization machinery. I factor transposition leads to its mRNA accumulating near and within the oocyte nucleus, thus causing perturbations in gurken and bicoid mRNA localization and axis specification. These observations further our understanding of the close association of transposable elements with their host and provide an explanation for how I factor transposition causes female sterility. We propose that the transposition of other elements may exploit the host's RNA transport signals and machinery.     

A bioinformatics search pipeline, RNA2DSearch, identifies RNA localization elements in Drosophila retrotransposons

mRNA localization is a widespread mode of delivering proteins to their site of function. The embryonic axes in Drosophila are determined in the oocyte, through Dynein-dependent transport of gurken/TGF-α mRNA, containing a small localization signal that assigns its destination. A signal with a similar secondary structure, but lacking significant sequence similarity, is present in the I factor retrotransposon mRNA, also transported by Dynein. It is currently unclear whether other mRNAs exist that are localized to the same site using similar signals. Moreover, searches for other genes containing similar elements have not been possible due to a lack of suitable bioinformatics methods for searches of secondary structure elements and the difficulty of experimentally testing all the possible candidates. We have developed a bioinformatics approach for searching across the genome for small RNA elements that are similar to the secondary structures of particular localization signals. We have uncovered 48 candidates, of which we were able to test 22 for their localization potential using injection assays for Dynein mediated RNA localization. We found that G2 and Jockey transposons each contain a gurken/I factor-like RNA stem–loop required for Dynein-dependent localization to the anterior and dorso–anterior corner of the oocyte. We conclude that I factor, G2, and Jockey are members of a “family” of transposable elements sharing a gurken-like mRNA localization signal and Dynein-dependent mechanism of transport. The bioinformatics pipeline we have developed will have broader utility in fields where small RNA signals play important roles.     

Nuclear import of CaMV P6 is required for infection and suppression of the RNA silencing factor DRB4

Replication of Cauliflower mosaic virus (CaMV), a plant double-stranded DNA virus, requires the viral translational transactivator protein P6. Although P6 is known to form cytoplasmic inclusion bodies (viroplasms) so far considered essential for virus biology, a fraction of the protein is also present in the nucleus. Here, we report that monomeric P6 is imported into the nucleus through two importin-alpha-dependent nuclear localization signals, and show that this process is mandatory for CaMV infectivity and is independent of translational transactivation and viroplasm formation. One nuclear function of P6 is to suppress RNA silencing, a gene regulation mechanism with antiviral roles, commonly counteracted by dedicated viral suppressor proteins (viral silencing suppressors; VSRs). Transgenic P6 expression in Arabidopsis is genetically equivalent to inactivating the nuclear protein DRB4 that facilitates the activity of the major plant antiviral silencing factor DCL4. We further show that a fraction of P6 immunoprecipitates with DRB4 in CaMV-infected cells. This study identifies both genetic and physical interactions between a VSR to a host RNA silencing component, and highlights the importance of subcellular compartmentalization in VSR function.     

Barentsz is essential for the posterior localization of oskar mRNA and colocalizes with it to the posterior pole

The localization of Oskar at the posterior pole of the Drosophila oocyte induces the assembly of the pole plasm and therefore defines where the abdomen and germ cells form in the embryo. This localization is achieved by the targeting of oskar mRNA to the posterior and the localized activation of its translation. oskar mRNA seems likely to be actively transported along microtubules, since its localization requires both an intact microtubule cytoskeleton and the plus end-directed motor kinesin I, but nothing is known about how the RNA is coupled to the motor. Here, we describe barentsz, a novel gene required for the localization of oskar mRNA. In contrast to all other mutations that disrupt this process, barentsz-null mutants completely block the posterior localization of oskar mRNA without affecting bicoid and gurken mRNA localization, the organization of the microtubules, or subsequent steps in pole plasm assembly. Surprisingly, most mutant embryos still form an abdomen, indicating that oskar mRNA localization is partially redundant with the translational control. Barentsz protein colocalizes to the posterior with oskar mRNA, and this localization is oskar mRNA dependent. Thus, Barentsz is essential for the posterior localization of oskar mRNA and behaves as a specific component of the oskar RNA transport complex.     

Evidence for common machinery utilized by the early and late RNA localization pathways in Xenopus oocytes

In Xenopus, an early and a late pathway exist for the selective localization of RNAs to the vegetal cortex during oogenesis. Previous work has suggested that distinct cellular mechanisms mediate localization during these pathways. Here, we provide several independent lines of evidence supporting the existence of common machinery for RNA localization during the early and late pathways. Data from RNA microinjection assays show that early and late pathway RNAs compete for common localization factors in vivo, and that the same short RNA sequence motifs are required for localization during both pathways. In addition, quantitative filter binding assays demonstrate that the late localization factor Vg RBP/Vera binds specifically to several early pathway RNA localization elements. Finally, confocal imaging shows that early pathway RNAs associate with a perinuclear microtubule network that connects to the mitochondrial cloud of stage I oocytes suggesting that motor driven transport plays a role during the early pathway as it does during the late pathway. Taken together, our data indicate that common machinery functions during the early and late pathways. Thus, RNA localization to the vegetal cortex may be a regulated process such that differential interactions with basal factors determine when distinct RNAs are localized during oogenesis.     

The E. coli RhlE RNA helicase regulates the function of related RNA helicases during ribosome assembly

Escherichia coli contains five members of the DEAD-box RNA helicase family, a ubiquitous class of proteins characterized by their ability to unwind RNA duplexes. Although four of these proteins have been implicated in RNA turnover or ribosome biogenesis, no cellular function for the RhlE DEAD-box protein has been described as yet. During an analysis of the cold-sensitive growth defect of a strain lacking the DeaD/CsdA RNA helicase, rhlE plasmids were identified from a chromosomal library as multicopy suppressors of the growth defect. Remarkably, when tested for allele specificity, RhlE overproduction was found to exacerbate the cold-sensitive growth defect of a strain that lacks the SrmB RNA helicase. Moreover, the absence of RhlE exacerbated or alleviated the cold-sensitive defect of deaD or srmB strains, respectively. Primer extension and ribosome analysis indicated that RhlE regulates the accumulation of immature ribosomal RNA or ribosome precursors when deaD or srmB strains are grown at low temperatures. By using an epitope-tagged version of RhlE, the majority of RhlE in cell extracts was found to cosediment with ribosome-containing fractions. Since both DeaD and SrmB have been recently shown to function in ribosome assembly, these findings suggests that rhlE genetically interacts with srmB and deaD to modulate their function during ribosome maturation. On the basis of the available evidence, I propose that RhlE is a novel ribosome assembly factor, which plays a role in the interconversion of ribosomal RNA-folding intermediates that are further processed by DeaD or SrmB during ribosome maturation.     

Native mitochondrial RNA-binding complexes in kinetoplastid RNA editing differ in guide RNA composition

Mitochondrial mRNAs in kinetoplastids require extensive U-insertion/deletion editing that progresses 3′-to-5′ in small blocks, each directed by a guide RNA (gRNA), and exhibits substrate and developmental stage-specificity by unsolved mechanisms. Here, we address compositionally related factors, collectively known as the mitochondrial RNA-binding complex 1 (MRB1) or gRNA-binding complex (GRBC), that contain gRNA, have a dynamic protein composition, and transiently associate with several mitochondrial factors including RNA editing core complexes (RECC) and ribosomes. MRB1 controls editing by still unknown mechanisms. We performed the first next-generation sequencing study of native subcomplexes of MRB1, immunoselected via either RNA helicase 2 (REH2), that binds RNA and associates with unwinding activity, or MRB3010, that affects an early editing step. The particles contain either REH2 or MRB3010 but share the core GAP1 and other proteins detected by RNA photo-crosslinking. Analyses of the first editing blocks indicate an enrichment of several initiating gRNAs in the MRB3010-purified complex. Our data also indicate fast evolution of mRNA 3′ ends and strain-specific alternative 3′ editing within 3′ UTR or C-terminal protein-coding sequence that could impact mitochondrial physiology. Moreover, we found robust specific copurification of edited and pre-edited mRNAs, suggesting that these particles may bind both mRNA and gRNA editing substrates. We propose that multiple subcomplexes of MRB1 with different RNA/protein composition serve as a scaffold for specific assembly of editing substrates and RECC, thereby forming the editing holoenzyme. The MRB3010-subcomplex may promote early editing through its preferential recruitment of initiating gRNAs.     

A complex RNA motif defined by three discontinuous 5-nucleotide-long strands is essential for Flavivirus RNA replication

Tertiary or higher-order RNA motifs that regulate replication of positive-strand RNA viruses are as yet poorly understood. Using Japanese encephalitis virus (JEV), we now show that a key element in JEV RNA replication is a complex RNA motif that includes a string of three discontinuous complementary sequences (TDCS). The TDCS consists of three 5-nt-long strands, the left (L) strand upstream of the translation initiator AUG adjacent to the 5′-end of the genome, and the middle (M) and right (R) strands corresponding to the base of the Flavivirus-conserved 3′ stem–loop structure near the 3′-end of the RNA. The three strands are arranged in an antiparallel configuration, with two sets of base-pairing interactions creating L-M and M-R duplexes. Disrupting either or both of these duplex regions of TDCS completely abolished RNA replication, whereas reconstructing both duplex regions, albeit with mutated sequences, fully restored RNA replication. Modeling of replication-competent genomes recovered from a large pool of pseudorevertants originating from six replication-incompetent TDCS mutants suggests that both duplex base-pairing potentials of TDCS are required for RNA replication. In all cases, acquisition of novel sequences within the 3′M-R duplex facilitated a long-range RNA–RNA interaction of its 3′M strand with either the authentic 5′L strand or its alternative (invariably located upstream of the 5′ initiator), thereby restoring replicability. We also found that a TDCS homolog is conserved in other flaviviruses. These data suggest that two duplex base-pairings defined by the TDCS play an essential regulatory role in a key step(s) of Flavivirus RNA replication.     

RNA localization in Xenopus oocytes uses a core group of trans-acting factors irrespective of destination

The 3′ untranslated region of mRNA encoding PHAX, a phosphoprotein required for nuclear export of U-type snRNAs, contains cis-acting sequence motifs E2 and VM1 that are required for localization of RNAs to the vegetal hemisphere of Xenopus oocytes. However, we have found that PHAX mRNA is transported to the opposite, animal, hemisphere. A set of proteins that cross-link to the localization elements of vegetally localized RNAs are also cross-linked to PHAX and An1 mRNAs, demonstrating that the composition of RNP complexes that form on these localization elements is highly conserved irrespective of the final destination of the RNA. The ability of RNAs to bind this core group of proteins is correlated with localization activity. Staufen1, which binds to Vg1 and VegT mRNAs, is not associated with RNAs localized to the animal hemisphere and may determine, at least in part, the direction of RNA movement in Xenopus oocytes.     

Functional characterization of the Drosophila MRP (mitochondrial RNA processing) RNA gene

MRP RNA is a noncoding RNA component of RNase mitochondrial RNA processing (MRP), a multi-protein eukaryotic endoribonuclease reported to function in multiple cellular processes, including ribosomal RNA processing, mitochondrial DNA replication, and cell cycle regulation. A recent study predicted a potential Drosophila ortholog of MRP RNA (CR33682) by computer-based genome analysis. We have confirmed the expression of this gene and characterized the phenotype associated with this locus. Flies with mutations that specifically affect MRP RNA show defects in growth and development that begin in the early larval period and end in larval death during the second instar stage. We present several lines of evidence demonstrating a role for Drosophila MRP RNA in rRNA processing. The nuclear fraction of Drosophila MRP RNA localizes to the nucleolus. Further, a mutant strain shows defects in rRNA processing that include a defect in 5.8S rRNA processing, typical of MRP RNA mutants in other species, as well as defects in early stages of rRNA processing.