Importance of translation and nonnucleolytic ago proteins for on-target RNA interference.

In animals, both siRNAs and miRNAs are thought to diminish protein synthesis from transcripts that are perfectly complementary by directing endonucleolytic cleavage where they anneal, thereby triggering rapid degradation of the entire message [1-4]. By contrast, partially complementary messages are downregulated by a combination of translational repression and accelerated decay caused by rapid poly(A) tail removal [3, 5-12]. Here we present evidence that translational repression can also make a substantial contribution to the downregulation of fully complementary messages by RNA interference. Unlike mRNA destabilization, this inhibitory effect on translation is greater for perfectly complementary elements located in the 3' untranslated region rather than in the protein-coding region. In addition to known disparities in their endonucleolytic activity [13, 14], the four Ago proteins with which siRNAs associate in humans differ significantly in their capacity to direct translational repression. As a result, the relative effect of siRNA on targets that are fully versus partially complementary is influenced by the comparative abundance of the three nonnucleolytic Ago proteins, causing this on-target/off-target ratio to vary in a cell-type-dependent manner because of the dissimilar tissue distribution of these proteins. These findings have important implications for the efficacy and specificity of RNA interference.

A specific cis-hairpin ribozyme facilitates infection of a TMV-based DNA vector in tobacco protoplasts.

The effect of a specific cis-hairpin ribozyme on TMV-based vectors in the infection of tobacco protoplasts was studied. Vectors contained full-length TMV genome cDNA linked to a T7 promoter or a CaMV 35S promoter at the 5'-end and an NOS gene polyadenylation signal at the 3'-end. The coat protein (CP) gene was replaced with the green fluorescent protein (GFPuv) gene allowing quantification of protoplast infection. In plasmids pTMVGFPRIB (T7-driven) and pSTMVGFPRIB (CaMV 35S-driven), the cDNA fragment of the cis-hairpin ribozyme (designed to specifically cleave the transcripts immediately downstream of the 3'-terminus of TMV RNA) was inserted between the 3'-terminus of TMV genome and NOS sequence. The in vitro transcript TMVGFPRIB was three- to fivefold more infectious than the control TMVGFPNOS. Northern blot analysis indicated that the 3'-terminal non-viral sequence had been cleaved from the in vitro transcripts by the cis-hairpin ribozyme soon after in vitro transcription. pSTMVGFPRIB and pSTMVGFPNOS plasmid DNAs were, as expected, less infectious than their in vitro transcript counterparts. However, pSTMVGFPRIB was somewhat more infectious than pSTMVGFPNOS. Northern blot analysis indicated that pSTMVGFPRIB synthesized more genomic and sub-genomic RNAs in the protoplasts. The significant increase in infectivity and viral RNA synthesis is due to the specific activity of the cis-hairpin ribozyme in vivo. Therefore, the cis-hairpin ribozyme described here may improve TMV-based vectors in the expression of foreign protein in plants.

Allele-specific targeting of microRNAs to HLA-G and risk of asthma.

HLA-G is a nonclassic, class I HLA molecule that has important immunomodulatory properties. Previously, we identified HLA-G as an asthma-susceptibility gene and discovered that the risk of asthma in a child was determined by both the child's HLA-G genotype and the mother's affection status. Here we report a SNP in the 3' untranslated region of HLA-G that influences the targeting of three microRNAs (miRNAs) to this gene, and we suggest that allele-specific targeting of these miRNAs accounts, at least in part, for our earlier observations on HLA-G and the risk of asthma.

MicroRNAs direct rapid deadenylation of mRNA.

MicroRNAs (miRNAs) are ubiquitous regulators of eukaryotic gene expression. In addition to repressing translation, miRNAs can down-regulate the concentration of mRNAs that contain elements to which they are imperfectly complementary. Using miR-125b and let-7 as representative miRNAs, we show that in mammalian cells this reduction in message abundance is a consequence of accelerated deadenylation, which leads to rapid mRNA decay. The ability of miRNAs to expedite poly(A) removal does not result from decreased translation; nor does translational repression by miRNAs require a poly(A) tail, a 3' histone stem-loop being an effective substitute. These findings suggest that miRNAs use two distinct posttranscriptional mechanisms to down-regulate gene expression.

CD24 is expressed by myofiber synaptic nuclei and regulates synaptic transmission.

The genes encoding several synaptic proteins, including acetylcholine receptors, acetylcholinesterase, and the muscle-specific kinase, MuSK, are expressed selectively by a small number of myofiber nuclei positioned near the synaptic site. Genetic analysis of mutant mice suggests that additional genes, expressed selectively by synaptic nuclei, might encode muscle-derived retrograde signals that regulate the differentiation of motor axon terminals. To identify candidate retrograde signals, we used a microarray screen to identify genes that are preferentially expressed in the synaptic region of muscle, and we analyzed one such gene, CD24, further. We show that CD24, which encodes a small, variably and highly glycosylated, glycosylphosphatidylinositol (GPI)-linked protein, is expressed preferentially by myofiber synaptic nuclei in embryonic and adult muscle, and that CD24 expression is restricted to the central region of muscle independent of innervation. Moreover, we show that CD24 has a role in presynaptic differentiation, because synaptic transmission is depressed and fails entirely, in a cyclical manner, after repetitive stimulation of motor axons in CD24 mutant mice. These deficits in synaptic transmission, which are accompanied by aberrant stimulus-dependent uptake of AM1-43 from axons, indicate that CD24 is required for normal presynaptic maturation and function. Because CD24 is also expressed in some neurons, additional experiments will be required to determine whether pre- or postsynaptic CD24 mediates these effects on presynaptic development and function.

A modified TMV-based vector facilitates the expression of longer foreign epitopes in tobacco.

Based upon a mutant isolated from tobacco infected with a recombinant tobacco mosaic virus (TMV), a new TMV-based vector was developed in which four to six C-terminal amino acid residues were deleted from the viral coat protein (CP) subunit. The new vector was quite similar to the original TMV-based vector, which all expressed a well characterized epitope peptide F11 (P(142)-A(152)) of VP1 from foot-and-mouth disease virus (FMDV) serotype O in tobacco, in the infectivity, yield of the virus particles and more importantly protective activity of F11 in guinea pigs and swine against the FMDV. Furthermore, the capacity of the length of foreign peptide encoded by this new vector was much improved to successfully express a peptide F25 containing two fused epitopes F14 (R(200)-L(213)) and F11 of FMDV VP1, which was failed using the original vector in tobacco. Although animal assays indicated that such expressed F25 was not as efficient as F11 in the immunity, possibly due to lack of a spacer arm between the two fused epitopes, the new TMV-based vector may meet the requirement of expressing longer foreign peptides for different vaccines and other medicines.

Runx1 prevents wasting, myofibrillar disorganization, and autophagy of skeletal muscle.

Disruptions in the use of skeletal muscle lead to muscle atrophy. After short periods of disuse, muscle atrophy is reversible, and even after prolonged periods of inactivity, myofiber degeneration is uncommon. The pathways that regulate atrophy, initiated either by peripheral nerve damage, immobilization, aging, catabolic steroids, or cancer cachexia, however, are poorly understood. Previously, we found that Runx1 (AML1), a DNA-binding protein that is homologous to Drosophila Runt and has critical roles in hematopoiesis and leukemogenesis, is poorly expressed in innervated muscle, but strongly induced in muscle shortly after denervation. To determine the function of Runx1 in skeletal muscle, we generated mice in which Runx1 was selectively inactivated in muscle. Here, we show that Runx1 is required to sustain muscle by preventing denervated myofibers from undergoing myofibrillar disorganization and autophagy, structural defects found in a variety of congenital myopathies. We find that only 29 genes, encoding ion channels, signaling molecules, and muscle structural proteins, depend upon Runx1 expression, suggesting that their misregulation causes the dramatic muscle wasting. These findings demonstrate an unexpected role for electrical activity in regulating muscle wasting, and indicate that muscle disuse induces compensatory mechanisms that limit myofiber atrophy. Moreover, these results suggest that reduced muscle activity could cause or contribute to congenital myopathies if Runx1 or its target genes were compromised.

Expression of foot-and-mouth disease virus epitopes in tobacco by a tobacco mosaic virus-based vector.

We expressed two immunogenic dominant epitopes of foot-and-mouth disease virus (FMDV) serotype O in tobacco plant using a vector based on a recombinant tobacco mosaic virus (TMV). The recombinant viruses TMVF11 and TMVF14 contained peptides of 11 and 14 amino acid residues, respectively, from FMDV VP 1 fused to the open reading frame of TMV coat protein (CP) gene between amino acid residues 154 and 155. TMVF11 and TMVF14 systemically infected tobacco plant and produced large quantities of stable progeny viral particles assembled with the modified CP subunits. Guinea pigs, mice and swine were used to test the protective effects of the recombinant viruses against FMDV infection. Most guinea pigs were protected against FMDV challenge after parenteral injection with TMVF11, TMVF14, or the mixture TMVF11/TMVF14, but not wtTMV. The TMVF11/TMVF14 mixture protected all animals when challenged with 150 guinea pig 50% infection dosage (GPID(50)) FMDV. Oral administration of the TMVF11/TMVF14 mixture (3mg total) protected 3/8 guinea pigs against the same FMDV challenge. Most of the suckling mice parenterally injected with antiserum from guinea pigs immunized with the TMVF11/TMVF14 mixture, but not with wtTMV, were also protected against FMDV challenge with 10 suckling mouse 50% lethal dosage (SMLD(50)), indicating that antibodies produced in guinea pigs immunized with the TMVF11/TMVF14 mixture specifically neutralized FMDV. Western blot analysis indicated that antiserum from those guinea pigs reacted with the FMDV VP1 protein. The protective effect of TMVF11 was also demonstrated in swine, where preliminary tests showed that nine pigs immunized with TMVF11 in three experiments were protected against FMDV challenge with 20 minimal infecting dose (MID).