Gemin3 is a DEAD-box RNA helicase that binds to the Survival of Motor Neurons (SMN) protein and is a component of the SMN complex, which also comprises SMN, Gemin2, Gemin4, Gemin5, and Gemin6. Reduction in SMN protein results in Spinal muscular atrophy (SMA), a common neurodegenerative disease. The SMN complex has critical functions in the assembly/restructuring of diverse ribonucleoprotein (RNP) complexes. Here we report that Gemin3 and Gemin4 are also in a separate complex that contains eIF2C2, a member of the Argonaute protein family. This novel complex is a large approximately 15S RNP that contains numerous microRNAs (miRNAs). We describe 40 miRNAs, a few of which are identical to recently described human miRNAs, a class of small endogenous RNAs. The genomic sequences predict that miRNAs are likely to be derived from larger precursors that have the capacity to form stem-loop structures.

Spinal muscular atrophy (SMA) is a common motor neuron degenerative disease that results from reduced levels of, or mutations in, the Survival of Motor Neurons (SMN) protein. SMN is found in the cytoplasm and the nucleus where it is concentrated in gems. SMN interacts with spliceosomal snRNP proteins and is critical for snRNP assembly in the cytoplasm. We show that a dominant-negative mutant SMN (SMNdeltaN27) causes a dramatic reorganization of snRNPs in the nucleus. Furthermore, SMNdeltaN27 inhibits pre-mRNA splicing in vitro, while wild-type SMN stimulates splicing. SMN mutants found in SMA patients cannot stimulate splicing. These findings demonstrate that SMN plays a crucial role in the generation of the pre-mRNA splicing machinery and thus in mRNA biogenesis, and they link the function of SMN in this pathway to SMA.

Spinal muscular atrophy (SMA) is a common motor neuron degenerative disease and the leading genetic cause of death of young children. The survival of motor neurons (SMN) gene, the SMA disease gene, is homozygously deleted or mutated in more than 98% of SMA patients. The SMN protein interacts with itself, with SMN-interacting protein 1, and with several spliceosomal small nuclear ribonucleoprotein (snRNP) Sm proteins. A complex containing SMN plays a critical role in spliceosomal snRNP assembly and in pre-mRNA splicing. SMN mutants found in SMA patients show reduced self-association and lack the capacity to regenerate the splicing machinery. Here we demonstrate that SMN mutants found in SMA patients are defective in binding to Sm proteins. Moreover, we show that SMN, but not mutants found in SMA patients, can form large oligomers and that SMN oligomerization is required for high-affinity binding to spliceosomal snRNP Sm proteins. These findings directly link the impaired interaction between SMN and Sm proteins to a defect in snRNP metabolism and to SMA.Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by the degeneration of motor neurons in the spinal cord, resulting in muscular weakness and atrophy. According to the severity and the age of onset of the disease, SMA patients are classified into three types: type I (WerdnigHoffmann disease), the most severe lethal form; type II, the intermediate form; and type III, the mildest form (1). The survival of motor neurons (SMN) gene has been identified as the disease gene of SMA, and two inverted SMN gene copies are present on human chromosome 5 at 5q13 (2-4). Only homozygous deletions or mutations of the SMN telomeric copy (SMN1) result in the SMA phenotype, and the levels of SMN expression driven by the centromeric copy (SMN2) in motor neurons inversely correlate with the severity of the disease (5, 6). The SMN2 gene, which does not provide complete protection from SMA, produces mainly an alternatively spliced form of SMN lacking exon 7 whose ratio, compared with the full length, also correlates with SMA severity (4,7,8).SMN and its associated protein SIP1 (SMN-interacting protein 1) are localized both in the cytoplasm and in the nucleus, where they concentrate in discrete bodies called gems (9, 10). SMN binds to itself, to SIP1, and to some of the spliceosomal small nuclear ribonucleoprotein (snRNP) Sm proteins (9-11). The interaction of SMN with the Sm proteins is likely to be important for the functions of the SMN complex in the assembly of snRNPs in the cytoplasm (12, 13) and in the nuclear regeneration of snRNPs and spliceosomes (14,15). Consistent with such critical housekeeping functions, SMN is expressed in all tissues of mammalian organisms and the mouse SMN gene knock-out displays an embryonic lethal phenotype (16). The evolutionarily highly conserved YG box domain (17), spanning exons 6 and 7, is important for SMN binding to Sm proteins (12) and for SMN self-association (13). A number of SMA patients have been shown to ha...

Gut epithelial cells contact both commensal and pathogenic bacteria, and proper responses to these bacteria require a balance of positive and negative regulatory signals. In the Drosophila intestine, peptidoglycan-recognition proteins (PGRPs), including PGRP-LE, play central roles in bacterial recognition and activation of immune responses, including induction of the IMD-NF-κB pathway. We show that bacteria recognition is regionalized in the Drosophila gut with various functional regions requiring different PGRPs. Specifically, peptidoglycan recognition by PGRP-LE in the gut induces NF-κB-dependent responses to infectious bacteria but also immune tolerance to microbiota through upregulation of pirk and PGRP-LB, which negatively regulate IMD pathway activation. Loss of PGRP-LE-mediated detection of bacteria in the gut results in systemic immune activation, which can be rescued by overexpressing PGRP-LB in the gut. Together these data indicate that PGRP-LE functions as a master gut bacterial sensor that induces balanced responses to infectious bacteria and tolerance to microbiota.

Drosophila hemocytes have strong phagocytic capacities and produce antimicrobial peptides (AMPs). However, the precise role of blood cells during immune responses and developmental processes has only been studied using indirect means. To overcome this limitation, we generated plasmatocyte-depleted flies by specifically overexpressing the proapoptotic protein Hid into plasmatocytes. Unexpectedly, these plasmatocyte-depleted animals have a normal larval and pupal development and do not exhibit any obvious defect after birth. Remarkably, plasmatocyte-depleted adults show a strong susceptibility to infections by various microorganisms, although activation of systemic AMP gene transcription via the Toll and immune deficiency (IMD) pathways is wild-type. Our data show that this susceptibility, which correlates with overproliferation of bacteria, is likely due to the absence of phagocytosis. We also demonstrate that during larval stages, plasmatocytes play an essential role in mediating AMP production by the fat body after oral bacterial infection. Finally, we show that plasmatocytes are involved in immune surveillance during pupal development, because they prevent bacterial infection that causes pupal lethality.AMP ͉ blood cells ͉ innate immunity ͉ Toll

Fat is an atypical cadherin that controls both cell growth and planar polarity. Atrophin is a nuclear co-repressor that is also essential for planar polarity; however, it is not known what genes Atrophin controls in planar polarity, or how Atrophin activity is regulated during the establishment of planar polarity. We show that Atrophin binds to the cytoplasmic domain of Fat and that Atrophin mutants show strong genetic interactions with fat. We find that both Atrophin and fat clones in the eye have non-autonomous disruptions in planar polarity that are restricted to the polar border of clones and that there is rescue of planar polarity defects on the equatorial border of these clones.Both fat and Atrophin are required to control four-jointed expression. In addition our mosaic analysis demonstrates an enhanced requirement for Atrophin in the R3 photoreceptor. These data lead us to a model in which fat and Atrophin act twice in the determination of planar polarity in the eye: first in setting up positional information through the production of a planar polarity diffusible signal, and later in R3 fate determination.

The survival motor neuron (SMN) protein, the protein product of the spinal muscular atrophy (SMA) disease gene, plays a role in the assembly and regeneration of small nuclear ribonucleoproteins (snRNPs) and spliceosomes. By nanoelectrospray mass spectrometry, we identified RNA helicase A (RHA) as an SMN complex–associated protein. RHA is a DEAH box RNA helicase which binds RNA polymerase II (pol II) and reportedly functions in transcription. SMN interacts with RHA in vitro, and this interaction is impaired in mutant SMNs found in SMA patients. Coimmunoprecipitation demonstrated that the SMN complex is associated with pol II, snRNPs, and RHA in vivo. In vitro experiments suggest that RHA mediates the association of SMN with the COOH-terminal domain of pol II. Moreover, transfection of cells with a dominant negative mutant of SMN, SMNΔN27, causes accumulation of pol II, snRNPs, and RHA in nuclear structures that contain the known markers of gems and coiled bodies, and inhibits RNA pol I and pol II transcription in vivo. These findings indicate a functional as well as physical association of the SMN complex with pol II and suggest a role for the SMN complex in the assembly of the pol II transcription/processing machinery.

These findings identify fibrillarin and GAR1 as novel interactors of SMN and suggest a function for the SMN complex in the assembly and metabolism of snoRNPs. We propose that the SMN complex performs functions necessary for the biogenesis and function of diverse ribonucleoprotein complexes.