Transgenic cassava resistance to African cassava mosaic virus is enhanced by viral DNA-A bidirectional promoter-derived siRNAs

Expression of double-stranded RNA (dsRNA) homologous to virus sequences can effectively interfere with RNA virus infection in plant cells by triggering RNA silencing. Here we applied this approach against a DNA virus, African cassava mosaic virus (ACMV), in its natural host cassava. Transgenic cassava plants were developed to express small interfering RNAs (siRNA) from a CaMV 35S promoter-controlled, intron-containing dsRNA cognate to the common region-containing bidirectional promoter of ACMV DNA-A. In two of three independent transgenic lines, accelerated plant recovery from ACMV-NOg infection was observed, which correlates with the presence of transgene-derived siRNAs 21-24nt in length. Overall, cassava mosaic disease symptoms were dramatically attenuated in these two lines and less viral DNA accumulation was detected in their leaves than in those of wild-type plants. In a transient replication assay using leaf disks from the two transgenic lines, strongly reduced accumulation of viral single-stranded DNA was observed. Our study suggests that a natural RNA silencing mechanism targeting DNA viruses through production of virus-derived siRNAs is turned on earlier and more efficiently in transgenic plants expressing dsRNA cognate to the viral promoter and common regio

Molecular characterization of geminivirus-derived small RNAs in different plant species

DNA geminiviruses are thought to be targets of RNA silencing. Here, we characterize small interfering (si) RNAs—the hallmarks of silencing—associated with Cabbage leaf curl begomovirus in Arabidopsis and African cassava mosaic begomovirus in Nicotiana benthamiana and cassava. We detected 21, 22 and 24 nt siRNAs of both polarities, derived from both the coding and the intergenic regions of these geminiviruses. Genetic evidence showed that all the 24 nt and a substantial fraction of the 22 nt viral siRNAs are generated by the dicer-like proteins DCL3 and DCL2, respectively. The viral siRNAs were 5′ end phosphorylated, as shown by phosphatase treatments, and methylated at the 3′-nucleotide, as shown by HEN1 miRNA methylase-dependent resistance to β-elimination. Similar modifications were found in all types of endogenous and transgene-derived siRNAs tested, but not in a major fraction of siRNAs from a cytoplasmic RNA tobamovirus. We conclude that several distinct silencing pathways are involved in DNA virus-plant interactions

Molecular characterization of geminivirus-derived small RNAs in different plant species

DNA geminiviruses are thought to be targets of RNA silencing. Here, we characterize small interfering (si) RNAs—the hallmarks of silencing—associated with Cabbage leaf curl begomovirus in Arabidopsis and African cassava mosaic begomovirus in Nicotiana benthamiana and cassava. We detected 21, 22 and 24 nt siRNAs of both polarities, derived from both the coding and the intergenic regions of these geminiviruses. Genetic evidence showed that all the 24 nt and a substantial fraction of the 22 nt viral siRNAs are generated by the dicer-like proteins DCL3 and DCL2, respectively. The viral siRNAs were 5′ end phosphorylated, as shown by phosphatase treatments, and methylated at the 3′-nucleotide, as shown by HEN1 miRNA methylase-dependent resistance to β-elimination. Similar modifications were found in all types of endogenous and transgene-derived siRNAs tested, but not in a major fraction of siRNAs from a cytoplasmic RNA tobamovirus. We conclude that several distinct silencing pathways are involved in DNA virus-plant interaction

Error-prone protein synthesis recapitulates early symptoms of Alzheimer disease in aging mice

Age-related neurodegenerative diseases (NDDs) are associated with the aggregation and propagation of specific pathogenic protein species (e.g., Aβ, α-synuclein). However, whether disruption of synaptic homeostasis results from protein misfolding per se rather than accumulation of a specific rogue protein is an unexplored question. Here, we show that error-prone translation, with its frequent outcome of random protein misfolding, is sufficient to recapitulate many early features of NDDs, including perturbed Ca2+ signaling, neuronal hyperexcitability, and mitochondrial dysfunction. Mice expressing the ribosomal ambiguity mutation Rps9 D95N exhibited disrupted synaptic homeostasis resulting in behavioral changes reminiscent of early Alzheimer disease (AD), such as learning and memory deficits, maladaptive emotional responses, epileptiform discharges, suppressed circadian rhythmicity, and sleep fragmentation, accompanied by hippocampal NPY expression and cerebral glucose hypometabolism. Collectively, our findings suggest that random protein misfolding may contribute to the pathogenesis of age-related NDDs, providing an alternative framework for understanding the initiation of AD. Keywords: Alzheimer; CP: Neuroscience; error-prone translation; neurodegenerative diseases; pathogenesis; protein misfolding; synaptic homeostasi

Analisis refleksi pada pembelajaran: review reasearch

Translation fidelity is the limiting factor in the accuracy of gene expression. With an estimated frequency of 10−4, errors in mRNA decoding occur in a mostly stochastic manner. Little is known about the response of higher eukaryotes to chronic loss of ribosomal accuracy as per an increase in the random error rate of mRNA decoding. Here, we present a global and comprehensive picture of the cellular changes in response to translational accuracy in mammalian ribosomes impaired by genetic manipulation. In addition to affecting established protein quality control pathways, such as elevated transcript levels for cytosolic chaperones, activation of the ubiquitin-proteasome system, and translational slowdown, ribosomal mistranslation led to unexpected responses. In particular, we observed increased mitochondrial biogenesis associated with import of misfolded proteins into the mitochondria and silencing of the unfolded protein response in the endoplasmic reticulum.ISSN:2399-364

Biochemical Requirements for Two Dicer-Like Activities from Wheat Germ

RNA silencing pathways were first discovered in plants. Through genetic analysis, it has been established that the key silencing components called Dicer-like (DCL) genes have been shown to cooperatively process RNA substrates of multiple origin into distinct 21, 22 and 24 nt small RNAs. However, only few detailed biochemical analysis of the corresponding complexes has been carried out in plants, mainly due to the large unstable complexes that are hard to obtain or reconstitute in heterologous systems. Reconstitution of activity needs thorough understanding of all protein partners in the complex, something that is still an ongoing process in plant systems. Here, we use biochemical analysis to uncover properties of two previously identified native dicer-like activities from wheat germ. We find that standard wheat germ extract contains Dicer-like enzymes that convert double-stranded RNA (dsRNA) into two classes of small interfering RNAs of 21 and 24 nt in size. The 21 nt dicing activity, likely an siRNA producing complex known as DCL4, is 950 kDa-1.2 mDa in size and is highly unstable during purification processes but has a rather vast range for activity. On the contrary, the 24 nt dicing complex, likely the DCL3 activity, is relatively stable and comparatively smaller in size, but has stricter conditions for effective processing of dsRNA substrates. While both activities could process completely complementary dsRNA albeit with varying abilities, we show that DCL3-like 24 nt producing activity is equally good in processing incompletely complementary RNAs

pH and temperature requirements.

<p><b>a</b>) Activity of DCL complexes across temperature range using S23 and individual DCL complexes. Range of temperature used is mentioned. Please refer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116736#pone.0116736.g001" target="_blank">Fig. 1</a> legend for other details. <b>b</b>) Activity of DCL complexes across a pH range using DCL activities.</p

Substrate processing abilities of DCL activities.

<p>Perfectly complementary (P) or imperfectly complementary (IMP; having a mismatch every 4^th base) substrates of the mentioned length were incubated with DCL activities to analyze their processing abilities. Note that 700 bp length substrate is perfectly complementary (Please see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116736#sec002" target="_blank">methods</a>).</p

Requirements of salt for DCL activities.

<p><b>a</b>) NaCl dependency of DCL activities. S23 extract was used to calculate optimal NaCl concentration required for DCL activities by providing labelled substrates. The extracts were first desalted by passing two times through sephadex G25 columns with buffers having 5 mM of the salt. Range of concentrations of NaCl used is shown. <b>b</b>) KAc dependency of DCL activities. Further details are similar to <b>a</b>.</p

Purification of native DCLs from wheat germ extracts.

<p><b>a</b>) Gel filtration analysis of crude wheat germ extract using Sephacryl S300. Fractions were collected and incubated with radiolabelled RNA substrates. The processed RNAs were precipitated with isopropanol, washed with 80% ethanol and dissolved in 10ul of loading dye (100% deionized formamide and 0.1% bromophenol blue) before separating on a 15% polyacrylamide gel. OD of the fractions was calculated separately and merged as green line with the gel picture. <b>b</b>) Determination of the sizes of DCL complexes. Three known marker proteins were used to draw a standard curve and sizes of DCL complexes was inferred using this standard curve. <b>c</b>) Ammonium sulfate precipitation to separate 24 and 21 nt generating activities. Please see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116736#sec002" target="_blank">methods</a> for details. Processed RNAs were detected as described above.</p