The soil nematodes Caenorhabditis briggsae and Caenorhabditis elegans diverged from a common ancestor roughly 100 million years ago and yet are almost indistinguishable by eye. They have the same chromosome number and genome sizes, and they occupy the same ecological niche. To explore the basis for this striking conservation of structure and function, we have sequenced the C. briggsae genome to a high-quality draft stage and compared it to the finished C. elegans sequence. We predict approximately 19,500 protein-coding genes in the C. briggsae genome, roughly the same as in C. elegans. Of these, 12,200 have clear C. elegans orthologs, a further 6,500 have one or more clearly detectable C. elegans homologs, and approximately 800 C. briggsae genes have no detectable matches in C. elegans. Almost all of the noncoding RNAs (ncRNAs) known are shared between the two species. The two genomes exhibit extensive colinearity, and the rate of divergence appears to be higher in the chromosomal arms than in the centers. Operons, a distinctive feature of C. elegans, are highly conserved in C. briggsae, with the arrangement of genes being preserved in 96% of cases. The difference in size between the C. briggsae (estimated at approximately 104 Mbp) and C. elegans (100.3 Mbp) genomes is almost entirely due to repetitive sequence, which accounts for 22.4% of the C. briggsae genome in contrast to 16.5% of the C. elegans genome. Few, if any, repeat families are shared, suggesting that most were acquired after the two species diverged or are undergoing rapid evolution. Coclustering the C. elegans and C. briggsae proteins reveals 2,169 protein families of two or more members. Most of these are shared between the two species, but some appear to be expanding or contracting, and there seem to be as many as several hundred novel C. briggsae gene families. The C. briggsae draft sequence will greatly improve the annotation of the C. elegans genome. Based on similarity to C. briggsae, we found strong evidence for 1,300 new C. elegans genes. In addition, comparisons of the two genomes will help to understand the evolutionary forces that mold nematode genomes.

The nematode worm Caenorhabditis elegans and its relatives are unique among animals in having operons. Operons are regulated multigene transcription units, in which polycistronic pre-messenger RNA (pre-mRNA coding for multiple peptides) is processed to monocistronic mRNAs. This occurs by 3' end formation and trans-splicing using the specialized SL2 small nuclear ribonucleoprotein particle for downstream mRNAs. Previously, the correlation between downstream location in an operon and SL2 trans-splicing has been strong, but anecdotal. Although only 28 operons have been reported, the complete sequence of the C. elegans genome reveals numerous gene clusters. To determine how many of these clusters represent operons, we probed full-genome microarrays for SL2-containing mRNAs. We found significant enrichment for about 1,200 genes, including most of a group of several hundred genes represented by complementary DNAs that contain SL2 sequence. Analysis of their genomic arrangements indicates that >90% are downstream genes, falling in 790 distinct operons. Our evidence indicates that the genome contains at least 1,000 operons, 2 8 genes long, that contain about 15% of all C. elegans genes. Numerous examples of co-transcription of genes encoding functionally related proteins are evident. Inspection of the operon list should reveal previously unknown functional relationships.

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Trans-splicing of one of two short leader RNAs, SL1 or SL2, occurs at the 59 ends of pre-mRNAs of many C. elegans genes. We have exploited RNA-sequencing data from the modENCODE project to analyze the transcriptome of C. elegans for patterns of trans-splicing. Transcripts of~70% of genes are trans-spliced, similar to earlier estimates based on analysis of far fewer genes. The mRNAs of most trans-spliced genes are spliced to either SL1 or SL2, but most genes are not transspliced to both, indicating that SL1 and SL2 trans-splicing use different underlying mechanisms. SL2 trans-splicing occurs in order to separate the products of genes in operons genome wide. Shorter intercistronic distance is associated with greater use of SL2. Finally, increased use of SL1 trans-splicing to downstream operon genes can indicate the presence of an extra promoter in the intercistronic region, creating what has been termed a ''hybrid'' operon. Within hybrid operons the presence of the two promoters results in the use of the two SL classes: Transcription that originates at the promoter upstream of another gene creates a polycistronic pre-mRNA that receives SL2, whereas transcription that originates at the internal promoter creates transcripts that receive SL1. Overall, our data demonstrate that >17% of all C. elegans genes are in operons.[Supplemental material is available for this article. The sequence data from this study have been submitted to the NCBI Sequence Read Archive (http://www.ncbi.nlm.nih.gov/Traces/sra/sra.cgi) under accession nos. SRA008646 and SRA003622.] C. elegans uses two RNA processing features that distinguish it from other model organisms. First, the transcripts of many genes are trans-spliced to a spliced leader (SL). Trans-splicing is a process in which an SL replaces the 59 end of a transcript by spliceosomal splicing. The 22-nucleotide (nt) SL is donated by an ;100-nt SL snRNP (small nuclear ribonucleoprotein) to a pre-mRNA with an intron-like region, the outron, containing an unpaired 39 splice site located near the 59 end. The second distinguishing feature is that many genes are transcribed in polycistronic units, known as operons, where a single promoter serves several genes. The operons can be up to eight genes long, and the polycistronic pre-mRNAs are separated into individual cistrons by 39 end formation accompanied by SL trans-splicing.These two features have important implications for C. elegans research. For instance, deletions/insertions within an operon may affect not only the expression of the gene containing the mutation, but also the genes downstream from it in the operon (Cui et al. 2008). Similarly, in a strain with enhanced RNAi sensitivity, RNAi of an operon gene can also affect expression of genes downstream (Guang et al. 2010). Finally, the trans-splice site is at the 59 end of the mRNA, not the pre-mRNA, and thus, the promoter is often not directly adjacent to the gene, but rather upstream of the outron or the entire operon (Blumenthal and Spieth 1996). SL trans-splicing has been repor...

Although eukaryotic genes are usually transcribed individually, at least a few Caenorhabditis elegans genes appear to be transcribed polycistronically in clusters resembling bacterial operons. The spliced leader SL2 (ref. 2) is specific for trans-splicing to downstream genes in these operons. In addition, many C. elegans pre-mRNAs are trans-spliced to SL1 (ref. 3) near the 5' ends of pre-mRNAs. Because operons have not previously been found in higher eukaryotes, we have investigated how widespread they are in the C. elegans genome. We identified gene clusters using the extensive data generated by the genome project and tested seven for trans-splicing specificity. All were found to fit expectations for polycistronic transcription. In addition, we surveyed reported C. elegans genes for trans-splicing specificity. Both methods indicate that the pre-mRNAs of about 70% of C. elegans genes are trans-spliced and as many as a quarter are transcribed in operons.

Introns are defined by sequences that bind components of the splicing machinery. The branchpoint consensus, polypyrimidine (poly(Y)) tract, and AG at the splice boundary comprise the mammalian 3' splice site. Although the AG is crucial for the recognition of introns with relatively short poly(Y) tracts, which are termed 'AG-dependent introns', the molecule responsible for AG recognition has never been identified. A key player in 3' splice site definition is the essential heterodimeric splicing factor U2AF, which facilitates the interaction of the U2 small nuclear ribonucleoprotein particle with the branch point. The U2AF subunit with a relative molecular mass (Mr 65K) of 65,000 (U2AF65) binds to the poly(Y) tract, whereas the role of the 35K subunit (U2AF35) has not been clearly defined. It is not required for splicing in vitro but it plays a critical role in vivo. Caenorhabditis elegans introns have a highly conserved U4CAG/ R at their 3' splice sites instead of branch-point and poly(Y) consensus sequences. Nevertheless, C. elegans has U2AF, 12). Here we show that both U2AF subunits crosslink to the 3' splice site. Our results suggest that the U2AF65-U2AF35 complex identifies the U4CAG/R, with U2AF35 being responsible for recognition of the canonical AG.

In the United States, estimates indicate there are between 250,000 and 400,000 individuals with Down syndrome (DS), and nearly all will develop Alzheimer's disease (AD) pathology starting in their 30s. With the current lifespan being 55 to 60 years, approximately 70% will develop dementia, and if their life expectancy continues to increase, the number of individuals developing AD will concomitantly increase. Pathogenic and mechanistic links between DS and Alzheimer's prompted the Alzheimer's Association to partner with the Linda Crnic Institute for Down Syndrome and the Global Down Syndrome Foundation at a workshop of AD and DS experts to discuss similarities and differences, challenges, and future directions for this field. The workshop articulated a set of research priorities: (1) target identification and drug development, (2) clinical and pathological staging, (3) cognitive assessment and clinical trials, and (4) partnerships and collaborations with the ultimate goal to deliver effective disease-modifying treatments.

Recognition of the 5-cap structure of mRNA by eIF4E is a critical step in the recruitment of most mRNAs to the ribosome. In Caenorhabditis elegans, ϳ70% of mRNAs contain an unusual 2,2,7-trimethylguanosine cap structure as a result of trans-splicing onto the 5 end of the pre-mRNA. The characterization of three eIF4E isoforms in C. elegans (IFE-1, IFE-2, and IFE-3) was reported previously. The present study describes two more eIF4E isoforms expressed in C. elegans, IFE-4 and IFE-5. We analyzed the requirement of each isoform for viability by RNA interference. IFE-3, the most closely related to mammalian eIF4E-1, binds only 7-methylguanosine caps and is essential for viability. In contrast, three closely related isoforms (IFE-1, IFE-2, and IFE-5) bind 2,2,7-trimethylguanosine caps and are partially redundant, but at least one functional isoform is required for viability. IFE-4, which binds only 7-methylguanosine caps, is most closely related to an unusual eIF4E isoform found in plants (nCBP) and mammals (4E-HP) and is not essential for viability in any combination of IFE knockout. ife-2, ife-3, ife-4, and ife-5 mRNAs are themselves trans-spliced to SL1 spliced leaders. ife-1 mRNA is transspliced to an SL2 leader, indicating that its gene resides in a downstream position of an operon.Eukaryotic mRNAs and small nuclear RNAs synthesized by RNA polymerase II are posttranscriptionally modified to form a 5Ј-5Ј GpppN linkage (1). The 5Ј-terminal G is methylated at N7 while still in the nucleus to yield an MMG 1 cap. The cap of small nuclear RNAs is then further methylated at N2 in the cytoplasm to yield a TMG cap (2). Methylation of small nuclear RNAs is dependent upon the binding of Sm proteins to form small nuclear ribonucleoproteins. Formation of the TMG cap is the targeting signal for import of small nuclear ribonucleoproteins back into the nucleus to take part in pre-mRNA splicing (3, 4). mRNAs, on the other hand, which possess only the MMG cap, remain in the cytoplasm.In some primative eukaryotes, including Caenorhabditis elegans, mRNAs acquire a TMG cap through the process of trans-splicing (5). Primary transcripts from approximately 70% of protein-coding genes are trans-spliced to 22-nt SL sequences, such that the original MMG caps are replaced with the TMG caps from the SL small nuclear RNAs (6, 7). Also common in C. elegans is the organization of genes into operons that are transcribed from a single promotor into a polycistronic RNA (8). trans-Splicing results in the processing of these primary transcripts into monocistronic mRNAs. Generally, the mRNA from the first cistron is trans-spliced to SL1,whereas mRNAs from downstream cistrons are trans-spliced to SL2 or SL2 variants (8). mRNAs that are not trans-spliced retain the original MMG cap. Thus, both MMG-and TMG-capped mRNAs are found in the cytoplasm of C. elegans. Both types of mRNA enter polyribosomes and are translated, indicating that they are competent to interact with the translational machinery (9).The recruitment of mRNAs to ribosomes is catalyzed by...