To identify components involved in the nuclear export of ribosomes in yeast, we developed an in vivo assay exploiting a green fluorescent protein (GFP)-tagged version of ribosomal protein L25. After its import into the nucleolus, L25-GFP assembles with 60S ribosomal subunits that are subsequently exported into the cytoplasm. In wild-type cells, GFP-labeled ribosomes are only detected by fluorescence in the cytoplasm. However, thermosensitive rna1-1 (Ran-GAP), prp20-1 (Ran-GEF), and nucleoporin nup49 and nsp1 mutants are impaired in ribosomal export as revealed by nuclear accumulation of L25-GFP. Furthermore, overexpression of dominant-negative RanGTP (Gsp1-G21V) and the tRNA exportin Los1p inhibits ribosomal export. The pattern of subnuclear accumulation of L25-GFP observed in different mutants is not identical, suggesting that transport can be blocked at different steps. Thus, nuclear export of ribosomes requires the nuclear/cytoplasmic Ran-cycle and distinct nucleoporins. This assay can be used to identify soluble transport factors required for nuclear exit of ribosomes.
Short interfering RNAs (siRNAs) are widely used as tool for gene inactivation in basic research and therapeutic applications. One of the major shortcomings of siRNA experiments are sequence-specific off-target effects. Such effects are largely unpredictable because siRNAs can affect partially complementary sequences and function like microRNAs (miRNAs), which inhibit gene expression on mRNA stability or translational levels. Here we demonstrate that novel, enzymatically generated siRNA pools—referred to as siPools—containing up to 60 accurately defined siRNAs eliminate off-target effects. This is achieved by the low concentration of each individual siRNA diluting sequence-specific off-target effects below detection limits. In fact, whole transcriptome analyses reveal that single siRNA transfections can severely affect global gene expression. However, when complex siRNA pools are transfected, almost no transcriptome alterations are observed. Taken together, we present enzymatically produced complex but accurately defined siRNA pools with potent on-target silencing but without detectable off-target effects.
SummaryThe gene devA of the filamentous heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 encodes a protein with high similarity to ATP-binding cassettes of ABC transporters. Mutant M7 defective in the devA gene is arrested in the development of heterocysts at an early stage and is not able to fix N 2 under aerobic conditions. The devA gene is differentially expressed in heterocysts. To gain a better understanding of the structural components of this putative ABC transporter, we determined the complete nucleotide sequence of the entire gene cluster. The two additional genes, named devB and devC, encode proteins with similarities to membrane fusion proteins (DevB) of several ABC exporters and to membrane-spanning proteins (DevC) of ABC transporters in general. Sitedirected mutations in each of the three genes resulted in identical phenotypes. Heterocyst-specific glycolipids forming the laminated layer of the envelope were identified in lipid extracts of M7 and in the site-directed mutants. However, transmission electron microscopy revealed unequivocally that the glycolipid layer is missing in mutant M7. Ultrastructural analysis also confirmed a developmental block at an early stage of differentiation. The results of this study suggest that the devBCA operon encodes an exporter of glycolipids or of an enzyme that is necessary for the formation of the laminated layer. The hypothesis is proposed that an intact envelope could be required for further heterocyst differentiation.
SMNrp, also termed SPF30, has recently been identi®ed in spliceosomes assembled in vitro. We have functionally characterized this protein and show that it is an essential splicing factor. We show that SMNrp is a 17S U2 snRNP-associated protein that appears in the pre-spliceosome (complex A) and the mature spliceosome (complex B) during splicing. Immunodepletion of SMNrp from nuclear extract inhibits the ®rst step of pre-mRNA splicing by preventing the formation of complex B. Re-addition of recombinant SMNrp to immunodepleted extract reconstitutes both spliceosome formation and splicing. Mutations in two domains of SMNrp, although similarly deleterious for splicing, differed in their consequences on U2 snRNP binding, suggesting that SMNrp may also engage in interactions with splicing factors other than the U2 snRNP. In agreement with this, we present evidence for an additional interaction between SMNrp and the [U4/U6´U5] tri-snRNP. A candidate that may mediate this interaction, namely the U4/U6-90 kDa protein, has been identi®ed. We suggest that SMNrp, as a U2 snRNP-associated protein, facilitates the recruitment of the [U4/U6´U5] tri-snRNP to the pre-spliceosome.
MicroRNAs (miRNAs) guide Argonaute (Ago) proteins to distinct target mRNAs leading to translational repression and mRNA decay. Ago proteins interact with a member of the GW protein family, referred to as TNRC6A-C in mammals, which coordinate downstream gene-silencing processes. The cytoplasmic functions of TNRC6 and Ago proteins are reasonably well established. Both protein families are found in the nucleus as well. Their detailed nuclear functions, however, remain elusive. Furthermore, it is not clear which import routes Ago and TNRC6 proteins take into the nucleus. Using different nuclear transport assays, we find that Ago as well as TNRC6 proteins shuttle between the cytoplasm and the nucleus. While import receptors might function redundantly to transport Ago2, we demonstrate that TNRC6 proteins are imported by the Importin-β pathway. Finally, we show that nuclear localization of both Ago2 and TNRC6 proteins can depend on each other suggesting actively balanced cytoplasmic Ago – TNRC6 levels.
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