Messenger RNAs containing premature stop codons are generally targeted for degradation through nonsense-mediated mRNA decay (NMD). This mechanism degrades aberrant transcripts derived from mutant genes containing nonsense or frameshift mutations. Wild-type genes also give rise to transcripts targeted by NMD. For example, some wild-type genes give rise to alternatively spliced transcripts that are targeted for decay by NMD. In Caenorhabditis elegans, the ribosomal protein (rp) L12 gene generates a nonsense codon-bearing alternatively spliced transcript that is induced in an autoregulatory manner by the rpL12 protein. By pharmacologically blocking the NMD pathway, we identified alternatively spliced mRNA transcripts derived from the human rpL3 and rpL12 genes that are natural targets of NMD. The deduced protein sequence of these alternatively spliced transcripts suggests that they are unlikely to encode functional ribosomal proteins. Overexpression of rpL3 increased the level of the alternatively spliced rpL3 mRNA and decreased the normally expressed rpL3. This indicates that rpL3 regulates its own production by a negative feedback loop and suggests the possibility that NMD participates in this regulatory loop by degrading the non-functional alternatively spliced transcript.

Human sex chromosomes, which are morphologically and genetically different, share few regions of homology. Among them, only pseudoautosomal regions (PARs) pair and recombine during meiosis. To better address the complex biology of these regions, we sequenced the telomeric 400 kb of the long arm of the human X chromosome, including 330 kb of the human Xq/YqPAR and the telomere. Sequencing reveals subregions with distinctive regulatory and evolutionary features. The proximal 295 kb contains two genes inactivated on both the inactive X and Y chromosomes [ SYBL1 and a novel homologue ( HSPRY3 ) of Drosophila sprouty ]. The GC-rich distal 35 kb, added in stages and much later in evolution, contains the X/Y expressed gene IL9R and a novel gene, CXYorf1, only 5 kb from the Xq telomere. These properties make Xq/YqPAR a model for studies of region-specific gene inactivation, telomere evolution, and involvement in sex-limited conditions.

The human ribosomal protein L7a is a component of the major ribosomal subunit. We previously identified three nuclear-localization-competent domains within L7a, and demonstrated that the domain defined by aa (amino acids) 52-100 is necessary, although not sufficient, to target the L7a protein to the nucleoli. We now demonstrate that L7a interacts in vitro with a presumably G-rich RNA structure, which has yet to be defined. We also demonstrate that the L7a protein contains two RNA-binding domains: one encompassing aa 52-100 (RNAB1) and the other encompassing aa 101-161 (RNAB2). RNAB1 does not contain any known nucleic-acid-binding motif, and may thus represent a new class of such motifs. On the other hand, a specific region of RNAB2 is highly conserved in several other protein components of the ribonucleoprotein complex. We have investigated the topology of the L7a-RNA complex using a recombinant form of the protein domain that encompasses residues 101-161 and a 30mer poly(G) oligonucleotide. Limited proteolysis and cross-linking experiments, and mass spectral analyses of the recombinant protein domain and its complex with poly(G) revealed the RNA-binding region.

mRNA localization is a conserved post-transcriptional process crucial for a variety of systems. We have analyzed the subcellular distribution of mRNAs encoding human cytosolic and mitochondrial ribosomal proteins. Biochemical fractionation experiments showed that the transcripts for cytosolic ribosomal proteins associate preferentially with the cytoskeleton via actin microfilaments. Transfection in HeLa cells of a GFP reporter construct containing the cytosolic ribosomal protein L4 3'-UTR showed that the 3'-UTR is necessary for the association of the transcript to the cytoskeleton. Using confocal analysis we demonstrate that the chimeric transcript is specifically associated with the perinuclear cytoskeleton. We also show that mRNA for mitochondrial ribosomal protein S12 is asymmetrically distributed in the cytoplasm. In fact, this transcript was localized mainly in the proximity of mitochondria, and the localization was 3'-UTR-dependent. In summary, ribosomal protein mRNAs constitute a new class of localized transcripts that share a common localization mechanism.

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