ABSTRACT
In eukaryotic cells, the biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) and likely other RNPs is mediated by an assemblyosome, the survival of motor neurons (SMN) complex. The SMN complex, composed of SMN and the Gemins (2-7), binds to the Sm proteins and to snRNAs and constructs the heptameric rings, the common cores of Sm proteins, on the Sm site (AU(56)G) of the snRNAs. We have determined the specific sequence and structural features of snRNAs for binding to the SMN complex and Sm core assembly. The minimal SMN complex-binding domain in snRNAs (except U1) is composed of an Sm site and a closely adjacent 3'stem-loop. Remarkably, the specific sequence of the stemloop is not important for SMN complex binding, but it must be located within a short distance of the 3'end of the RNA for an Sm core to assemble. This minimal snRNA-defining "snRNP code" is recognized by the SMN complex, which binds to it directly and with high affinity and assembles the Sm core. The recognition of the snRNAs is provided by Gemin5, a component of the SMN complex that directly binds the snRNP code. Gemin5 is a novel RNA-binding protein that is critical for snRNP biogenesis. Thus, the SMN complex is the identifier, as well as assembler, of the abundant class of snRNAs in cells. The function of the SMN complex, previously unanticipated because RNP biogenesis was believed to occur by self-assembly, confers stringent specificity on otherwise potentially illicit RNA-protein interactions.
Subject(s)
Cyclic AMP Response Element-Binding Protein/chemistry , Cyclic AMP Response Element-Binding Protein/metabolism , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , Ribonucleoproteins/chemistry , Ribonucleoproteins/metabolism , Base Sequence , HeLa Cells , Humans , Models, Biological , Molecular Sequence Data , Multiprotein Complexes , Nucleic Acid Conformation , RNA, Small Nuclear/biosynthesis , RNA, Small Nuclear/chemistry , RNA, Small Nuclear/genetics , Ribonucleoproteins, Small Nuclear/biosynthesis , Ribonucleoproteins, Small Nuclear/chemistry , Ribonucleoproteins, Small Nuclear/metabolism , SMN Complex Proteins , Spliceosomes/metabolismABSTRACT
Replication-dependent histone mRNAs end in a highly conserved 26-nt stem-loop structure. The stem-loop binding protein (SLBP), an evolutionarily conserved protein with no known homologs, interacts with the stem-loop in both the nucleus and cytoplasm and mediates nuclear-cytoplasmic transport as well as 3'-end processing of the pre-mRNA by the U7 snRNP. Here, we examined the affinity and specificity of the SLBP-RNA interaction. Nitrocellulose filter-binding experiments showed that the apparent equilibrium dissociation constant (Kd) between purified SLBP and the stem-loop RNA is 1.5 nM. Binding studies with a series of stem-loop variants demonstrated that conserved residues in the stem and loop, as well as the 5' and 3' flanking regions, are required for efficient protein recognition. Deletion analysis showed that 3 nt 5' of the stem and 1 nt 3' of the stem contribute to the binding energy. These data reveal that the high affinity complex between SLBP and the RNA involves sequence-specific contacts to the loop and the top of the stem, as well the base of the stem and its immediate flanking sequences. Together, these results suggest a novel mode of protein-RNA recognition that forms the core of a ribonucleoprotein complex central to the regulation of histone gene expression.
Subject(s)
Nucleic Acid Conformation , RNA, Messenger/chemistry , RNA, Messenger/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , Xenopus Proteins , mRNA Cleavage and Polyadenylation Factors , Animals , Base Sequence , Binding Sites , Calorimetry , Cell Line , Consensus Sequence , Histones/metabolism , Humans , Models, Molecular , Molecular Sequence Data , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Ribosomes/metabolism , Spodoptera , Thermodynamics , Transfection , XenopusABSTRACT
There are many practical problems in which it is required to detect and characterize hidden structures or remote objects by virtue of the scattered acoustic or electromagnetic fields they generate. It remains an open question, however, as to which reconstruction algorithms offer the most informative images for a given set of field measurements. Commonly used time-domain beamforming techniques, and their equivalent frequency-domain implementations, are conceptually simple and stable in the presence of noise, however, large proportions of missing measurements can quickly degrade the image quality. We apply a new algorithm based on the maximum entropy method (MEM) to the reconstruction of images from sparsely sampled coherent field data. The general principles and limitations of the new method are discussed in the framework of regularization theory, and the results of monostatic imaging experiments confirm that superior resolution and artifact suppression are obtained relative to a commonly used linear inverse filtering approach.
