Summary FMRP loss-of-function causes Fragile X Syndrome (FXS) and autistic features. FMRP is a polyribosome-associated neuronal RNA-binding protein, suggesting that it plays a key role in regulating neuronal translation, but there has been little consensus regarding either its RNA targets or mechanism of action. Here we use high throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP) to identify FMRP interactions with mouse brain polyribosomal mRNAs. FMRP interacts with the coding region of transcripts encoding pre- and postsynaptic proteins, and transcripts implicated in autism spectrum disorders (ASD). We developed a brain polyribosome-programmed translation system, revealing that FMRP reversibly stalls ribosomes specifically on its target mRNAs. Our results indicate that loss of a translational brake on the synthesis of a subset of synaptic proteins may contribute to FXS. In addition, they provide insight into the molecular basis of the cognitive and allied defects in FXS and ASD, and suggest multiple targets for clinical intervention.

Alternative RNA splicing greatly increases proteome diversity and may thereby contribute to tissue-specific functions. We carried out genome-wide quantitative analysis of alternative splicing using a custom Affymetrix microarray to assess the role of the neuronal splicing factor Nova in the brain. We used a stringent algorithm to identify 591 exons that were differentially spliced in the brain relative to immune tissues, and 6.6% of these showed major splicing defects in the neocortex of Nova2-/- mice. We tested 49 exons with the largest predicted Nova-dependent splicing changes and validated all 49 by RT-PCR. We analyzed the encoded proteins and found that all those with defined brain functions acted in the synapse (34 of 40, including neurotransmitter receptors, cation channels, adhesion and scaffold proteins) or in axon guidance (8 of 40). Moreover, of the 35 proteins with known interaction partners, 74% (26) interact with each other. Validating a large set of Nova RNA targets has led us to identify a multi-tiered network in which Nova regulates the exon content of RNAs encoding proteins that interact in the synapse.

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Fragile-X mental retardation is caused by loss of function of a single gene encoding the Fragile-X mental retardation protein, FMRP, an RNA-binding protein that harbors two KH-type and one RGG-type RNA-binding domains. Previous studies identified intramolecular G-quartet RNAs as high-affinity targets for the RGG box, but the relationship of RNA binding to FMRP function and mental retardation remains unclear. One severely affected patient harbors a missense mutation (I304N) within the second KH domain (KH2), and some evidence suggests this domain may be involved in the proposed role of FMRP in translational regulation. We now identify the RNA target for the KH2 domain as a sequence-specific element within a complex tertiary structure termed the FMRP kissing complex. We demonstrate that the association of FMRP with brain polyribosomes is abrogated by competition with the FMRP kissing complex RNA, but not by high-affinity G-quartet RNAs. We conclude that mental retardation associated with the I304N mutation, and likely the Fragile-X syndrome more generally, may relate to a crucial role for RNAs harboring the kissing complex motif as targets for FMRP translational regulation.[Keywords: Fragile-X mental retardation; FMRP; polyribosome; loop-loop pseudoknot; kissing complex; RNA; KH domain] Supplemental material is available at http://www.genesdev.org.

Fragile X mental retardation protein (FMRP) is an RNA binding protein encoded by the gene FMR1, whose expression is impaired in patients with fragile X mental retardation. The association of FMRP with polyribosomes in non-neural cell lines has previously suggested that FMRP is involved in translational regulation. However, the relevance of these studies to neuronal function has been questioned by the finding that FMRP in brain is not associated with polyribosomes, but is part of small ribonucleo-protein complexes that do not appear to include ribosomes. Here we optimize methods to analyze brain polyribosomes, allowing us to definitively demonstrate that FMRP forms complexes with cortical brain polyribosomes. Moreover, we demonstrate in neuroblastoma cells that the FMRP-polyribosome complexes are sensitive to puromycin, a drug that targets actively translating ribosomes. These data indicate that FMRP associates with functional polyribosomes in neurons.

a b s t r a c tThis paper presents an automatic building detection technique using LIDAR data and multispectral imagery. Two masks are obtained from the LIDAR data: a 'primary building mask' and a 'secondary building mask'. The primary building mask indicates the void areas where the laser does not reach below a certain height threshold. The secondary building mask indicates the filled areas, from where the laser reflects, above the same threshold. Line segments are extracted from around the void areas in the primary building mask. Line segments around trees are removed using the normalized difference vegetation index derived from the orthorectified multispectral images. The initial building positions are obtained based on the remaining line segments. The complete buildings are detected from their initial positions using the two masks and multispectral images in the YIQ colour system. It is experimentally shown that the proposed technique can successfully detect urban residential buildings, when assessed in terms of 15 indices including completeness, correctness and quality.

Automatic camera calibration via self-calibration with the aid of coded targets is now very much the norm in closerange photogrammetry. This is irrespective of whether the cameras to be calibrated are high-end metric, or the digital SLRs and consumer-grade models that are increasingly being employed for image-based 3D measurement. Automation has greatly simplified the calibration task, but there are real prospects that important camera calibration issues may be overlooked in what has become an almost black-box operation. This paper discusses the impact of a number of such issues, some of which relate to the functional model adopted for self-calibration, and others to practical aspects which need to be taken into account when pursuing optimal calibration accuracy and integrity. Issues discussed include interior orientation stability, calibration reliability, focal plane distortion, image point distribution, variation in lens distortion with image scale, colour imagery and chromatic aberration, and whether 3D object space control is warranted. By appreciating and accounting for these issues, users of automatic camera calibration will enhance the prospect of achieving an optimal recovery of scene-independent camera calibration parameters.Unfortunately, the vast majority of cameras utilised today for moderate-accuracy close-range 3D measurement were not designed with photogrammetric accuracy in mind, so whereas it is certainly true that off-the-shelf digital cameras ranging from inexpensive consumer-grade to high-end prosumer-grade digital SLRs can be readily applied to photogrammetric measurement, their metric performance is generally well below the level that would be anticipated if the cameras were indeed fully 'metric'. One could say that this is due to a shortcoming in calibration, but as this paper will endeavour to illustrate, it is not so much the process of automated calibration that is deficient, but the nature of the cameras and shortcomings in the imaging networks employed.

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