Deficiency of the Ribosomal Protein uL5 Leads to Significant Rearrangements of the Transcriptional and Translational Landscapes in Mammalian Cells

Protein uL5 (formerly called L11) is an integral component of the large (60S) subunit of the human ribosome, and its deficiency in cells leads to the impaired biogenesis of 60S subunits. Using RNA interference, we reduced the level of uL5 in HEK293T cells by three times, which caused an almost proportional decrease in the content of the fraction corresponding to 80S ribosomes, without a noticeable diminution in the level of polysomes. By RNA sequencing of uL5-deficient and control cell samples, which were those of total mRNA and mRNA from the polysome fraction, we identified hundreds of differentially expressed genes (DEGs) at the transcriptome and translatome levels and revealed dozens of genes with altered translational efficiency (GATEs). Transcriptionally up-regulated DEGs were mainly associated with rRNA processing, pre-mRNA splicing, translation and DNA repair, while down-regulated DEGs were genes of membrane proteins; the type of regulation depended on the GC content in the 3′ untranslated regions of DEG mRNAs. The belonging of GATEs to up-regulated and down-regulated ones was determined by the coding sequence length of their mRNAs. Our findings suggest that the effects observed in uL5-deficient cells result from an insufficiency of translationally active ribosomes caused by a deficiency of 60S subunits.

MicroRNA Zma-miR528 Versatile Regulation on Target mRNAs during Maize Somatic Embryogenesis

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the accumulation and translation of their target mRNAs through sequence complementarity. miRNAs have emerged as crucial regulators during maize somatic embryogenesis (SE) and plant regeneration. A monocot-specific miRNA, mainly accumulated during maize SE, is zma-miR528. While several targets have been described for this miRNA, the regulation has not been experimentally confirmed for the SE process. Here, we explored the accumulation of zma-miR528 and several predicted targets during embryogenic callus induction, proliferation, and plantlet regeneration using the maize cultivar VS-535. We confirmed the cleavage site for all tested zma-miR528 targets; however, PLC1 showed very low levels of processing. The abundance of zma-miR528 slightly decreased in one month-induced callus compared to the immature embryo (IE) explant tissue. However, it displayed a significant increase in four-month sub-cultured callus, coincident with proliferation establishment. In callus-regenerated plantlets, zma-miR528 greatly decreased to levels below those observed in the initial explant. Three of the target transcripts (MATE, bHLH, and SOD1a) showed an inverse correlation with the miRNA abundance in total RNA samples at all stages. Using polysome fractionation, zma-miR528 was detected in the polysome fraction and exhibited an inverse distribution with the PLC1 target, which was not observed at total RNA. Accordingly, we conclude that zma-miR528 regulates multiple target mRNAs during the SE process by promoting their degradation, translation inhibition or both.

The influence of exogenous spermidine on polysomes and RNase activity in wheat leaves under water stress conditions short communication

It was found that spermidine (5mM) delivered to wheat seedlings through their roots prior to water stress affected the state of polysomes and ribosomes under water stress conditions, which was manifested in stabilization of 80 S ribosomes and in reduction of polysome fraction. Besides, it was observed that pretreatement with spermidine decreases RNase activity in plants exposed to water stress.

Small-Subunit rRNA Processome Proteins Are Translationally Regulated during Differentiation of Trypanosoma cruzi

ABSTRACT We used differential display to select genes differentially expressed during differentiation of epimastigotes into metacyclic trypomastigotes in the protozoan parasite Trypanosoma cruzi. One of the selected clones had a sequence similar to that of the small-subunit (SSU) processome protein Sof1p, which is involved in rRNA processing. The corresponding T. cruzi protein, TcSof1, displayed a nuclear localization and is downregulated during metacyclogenesis. Heterologous RNA interference assays showed that depletion of this protein impaired growth but did not affect progression through the cell cycle, suggesting that ribosome synthesis regulation and the cell cycle are uncoupled in this parasite. Quantitative PCR (qPCR) assays of several SSU processome-specific genes in T. cruzi also showed that most of them were regulated posttranscriptionally. This process involves the accumulation of mRNA in the polysome fraction of metacyclic trypomastigotes, where TcSof1 cannot be detected. Metacyclic trypomastigote polysomes were purified and separated by sucrose gradient sedimentation. Northern blot analysis of the sucrose gradient fractions showed the association of TcSof1 mRNA with polysomes, confirming the qPCR data. The results suggest that the mechanism of regulation involves the blocking of translation elongation and/or termination.

Mapping the Ribosomal Exit Site of the Nascent Polypeptide Chain by Immune Electron Microscopy

Immune electron microscopy is providing information on the three dimensional locations of many functional sites on the ribosome. In particular, localization of the exit site of the newly synthesized protein chain is expected to provide information on the mechanisms of protein synthesis and on the nature of the ribosome-membrane association. Theoretically the nascent chains of the largest proteins could be visible as they leave the ribosome, but in practice they have not been observed. In order to map their exit site we have made antibodies against one particular enzyme and then applied immune electron microscopy to a polysome fraction enriched for that special protein.In a mutant of Escherichia coli constitutive for the enzyme beta-galac- tosidase, this enzyme accounts for approximately 25% of the total protein (1). Because of the high molecular weight of beta-galactosidase, a fraction of larger polysomes, further enriched in this emzyme, was obtained by differential centrifugation. When IgG antibodies against this enzyme are reacted with polysomes, two types of profiles are observed.