Trisomy 21 (T21) causes Down syndrome (DS), but the mechanisms by which T21 produces the different disease spectrum observed in people with DS are unknown. We recently identified an activated interferon response associated with T21 in human cells of different origins, consistent with overexpression of the four interferon receptors encoded on chromosome 21, and proposed that DS could be understood partially as an interferonopathy. However, the impact of T21 on systemic signaling cascades in living individuals with DS is undefined. To address this knowledge gap, we employed proteomics approaches to analyze blood samples from 263 individuals, 165 of them with DS, leading to the identification of dozens of proteins that are consistently deregulated by T21. Most prominent among these proteins are numerous factors involved in immune control, the complement cascade, and growth factor signaling. Importantly, people with DS display higher levels of many pro-inflammatory cytokines (e.g. IL-6, MCP-1, IL-22, TNF-α) and pronounced complement consumption, resembling changes seen in type I interferonopathies and other autoinflammatory conditions. Therefore, these results are consistent with the hypothesis that increased interferon signaling caused by T21 leads to chronic immune dysregulation, and justify investigations to define the therapeutic value of immune-modulatory strategies in DS.

Metazoan introns contain a polypyrimidine tract immediately upstream of the AG dinucleotide that defines the 3′ splice site. In the nematode Caenorhabditis elegans, 3′ splice sites are characterized by a highly conserved UUUUCAG/R octamer motif. While the conservation of pyrimidines in this motif is strongly suggestive of their importance in pre-mRNA splicing, in vivo evidence in support of this is lacking. In an N-ethyl-N-nitrosourea (ENU) mutagenesis screen in Caenorhabditis elegans, we have isolated a strain containing a point mutation in the octamer motif of a 3′ splice site in the daf-12 gene. This mutation, a single base T-to-G transversion at the -5 position relative to the splice site, causes a strong daf-12 loss-of-function phenotype by abrogating splicing. The resulting transcript is predicted to encode a truncated DAF-12 protein generated by translation into the retained intron, which contains an in-frame stop codon. Other than the perfectly conserved AG dinucleotide at the site of splicing, G at the –5 position of the octamer motif is the most uncommon base in C. elegans 3′ splice sites, occurring at closely paired sites where the better match to the splicing consensus is a few bases downstream. Our results highlight both the biological importance of the highly conserved –5 uridine residue in the C. elegans 3′ splice site octamer motif as well as the utility of using ENU as a mutagen to study the function of polypyrimidine tracts and other AU- or AT-rich motifs in vivo.

Transcription termination is mechanistically coupled to pre-mRNA 3' end formation to prevent transcription much beyond the gene 3' end. C. elegans, however, engages in polycistronic transcription of operons in which 3' end formation between genes is not accompanied by termination. We have performed RNA polymerase II (RNAPII) and CstF ChIP-seq experiments to investigate at a genome-wide level how RNAPII can transcribe through multiple poly-A signals without causing termination. Our data shows that transcription proceeds in some ways as if operons were composed of multiple adjacent single genes. Total RNAPII shows a small peak at the promoter of the gene cluster and a much larger peak at 3' ends. These 3' peaks coincide with maximal phosphorylation of Ser2 within the C-terminal domain (CTD) of RNAPII and maximal localization of the 3' end formation factor CstF. This pattern occurs at all 3' ends including those at internal sites in operons where termination does not occur. Thus the normal mechanism of 3' end formation does not always result in transcription termination. Furthermore, reduction of CstF50 by RNAi did not substantially alter the pattern of CstF64, total RNAPII, or Ser2 phosphorylation at either internal or terminal 3' ends. However, CstF50 RNAi did result in a subtle reduction of CstF64 binding upstream of the site of 3' cleavage, suggesting that the CstF50/CTD interaction may facilitate bringing the 3' end machinery to the transcription complex.

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. (C) 2015 The Alzheimer's Association. Published by Elsevier Inc. All rights reserved.

Nearly 15% of the ~20,000 C. elegans genes are contained in operons, multigene clusters controlled by a single promoter. The vast majority of these are of a type where the genes in the cluster are ~100 bp apart and the pre-mRNA is processed by 3' end formation accompanied by trans-splicing. A spliced leader, SL2, is specialized for operon processing. Here we summarize current knowledge on several variations on this theme including: (1) hybrid operons, which have additional promoters between genes; (2) operons with exceptionally long (> 1 kb) intercistronic regions; (3) operons with a second 3' end formation site close to the trans-splice site; (4) alternative operons, in which the exons are sometimes spliced as a single gene and sometimes as two genes; (5) SL1-type operons, which use SL1 instead of SL2 to trans-splice and in which there is no intercistronic space; (6) operons that make dicistronic mRNAs; and (7) non-operon gene clusters, in which either two genes use a single exon as the 3' end of one and the 5' end of the next, or the 3' UTR of one gene serves as the outron of the next. Each of these variations is relatively infrequent, but together they show a remarkable variety of tight-linkage gene arrangements in the C. elegans genome.

Caenorhabditis elegans mutants deleted for TDP-1, an ortholog of the neurodegeneration-associated RNA-binding protein TDP-43, display only mild phenotypes. Nevertheless, transcriptome sequencing revealed that many RNAs were altered in accumulation and/or processing in the mutant. Analysis of these transcriptional abnormalities demonstrates that a primary function of TDP-1 is to limit formation or stability of double-stranded RNA. Specifically, we found that deletion of tdp-1: (1) preferentially alters the accumulation of RNAs with inherent double-stranded structure (dsRNA); (2) increases the accumulation of nuclear dsRNA foci; (3) enhances the frequency of adenosine-to-inosine RNA editing; and (4) dramatically increases the amount of transcripts immunoprecipitable with a dsRNA-specific antibody, including intronic sequences, RNAs with antisense overlap to another transcript, and transposons. We also show that TDP-43 knockdown in human cells results in accumulation of dsRNA, indicating that suppression of dsRNA is a conserved function of TDP-43 in mammals. Altered accumulation of structured RNA may account for some of the previously described molecular phenotypes (e.g., altered splicing) resulting from reduction of TDP-43 function.

I do understand that you would prefer to develop the mechanism of dsRNA regulation by Tdp1 as a separate story; however, I am not fully convinced that the currently offered additional data would suffice for the referees to support publication (since all three referees specifically point to the lack of mechanistic insight as a major concern). An alternative option that may help sway the referees, would be for you to instead include the full data set showing enhanced RNAi in tdp1-depleted animals. Not only would this go well with the genetic data for chemotaxis in tdp1 mutants (and thus address two separate concerns raised by ref#1), it would also add important insight on the functional outcome of Tdp1 depletion, as requested by the referees. I would therefore strongly encourage you to include this data in the revised manuscript. With regard to the manuscript length, we do not impose limitations here and we would be happy to allow you the required space to develop the study as requested by the referees.

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.

More than half of Caenorhabditis elegans pre-mRNAs lose their original 5′ ends in a process termed “ trans -splicing” in which the RNA extending from the transcription start site (TSS) to the site of trans -splicing of the primary transcript, termed the “outron,” is replaced with a 22-nt spliced leader. This complicates the mapping of TSSs, leading to a lack of available TSS mapping data for these genes. We used growth at low temperature and nuclear isolation to enrich for transcripts still containing outrons, applying a modified SAGE capture procedure and high-throughput sequencing to characterize 5′ termini in this transcript population. We report from this data both a landscape of 5′-end utilization for C. elegans and a representative collection of TSSs for 7351 trans -spliced genes. TSS distributions for individual genes were often dispersed, with a greater average number of TSSs for trans -spliced genes, suggesting that trans -splicing may remove selective pressure for a single TSS. Upstream of newly defined TSSs, we observed well-known motifs (including TATAA-box and SP1) as well as novel motifs. Several of these motifs showed association with tissue-specific expression and/or conservation among six worm species. Comparing TSS features between trans -spliced and non- trans -spliced genes, we found stronger signals among outron TSSs for preferentially positioning of flanking nucleosomes and for downstream Pol II enrichment. Our data provide an enabling resource for both experimental and theoretical analysis of gene structure and function in C. elegans .

About 70% of C. elegans mRNAs are trans-spliced to one of two 22 nucleotide spliced leaders. SL1 is used to trim off the 5' ends of pre-mRNAs and replace them with the SL1 sequence. This processing event is very closely related to cis-splicing, or intron removal. The SL1 sequence is donated by a 100 nt small nuclear ribonucleoprotein particle (snRNP). This snRNP is structurally and functionally related to the U snRNAs (U1, U2, U4, U5 and U6) that play key roles in intron removal and trans-splicing, except that it is consumed in the process of splicing. More than half of C. elegans pre-mRNAs are subject to SL1 trans-splicing. About 30% are not trans-spliced at all. The remaining genes are trans-spliced by SL2. These genes are all downstream genes in closely spaced gene clusters similar to bacterial operons. They are transcribed from a promoter at the 5' end of the cluster of between 2 and 8 genes. This transcription makes a polycistronic pre-mRNA that is co-transcriptionally processed by cleavage and polyadenylation at the 3' end of each gene, and this event is closely coupled to the SL2 trans-splicing event that occurs only approximately 100 nt further downstream. Recent studies on the mechanism of SL2 trans-splicing have revealed that one of the 3' end formation proteins, CstF, interacts with the only protein known to be specific to the SL2 snRNP. The operons contain primarily genes whose products are needed for mitochondrial function and the basic machinery of gene expression: transcription, splicing and translation. Many operons contain genes whose products are known to function together. This presumably provides co-regulation of these proteins by producing a single RNA that encodes both.

Some genes in the candidate early-branching eukaryote Giardia lamblia occur in separate pieces, transcribed from non-contiguous chromosomal locations. The pre-mRNAs from the separate pieces apparently find each other by regions of complementarity and are subsequently spliced together by the spliceosome. Could genes in pieces, transcribed into separate pre-mRNAs, have been an early feature of spliceosomal evolution?

Trans-splicing is the joining together of portions of two separate pre-mRNA molecules. The two distinct categories of spliceosomal trans-splicing are genic trans-splicing, which joins exons of different pre-mRNA transcripts, and spliced leader (SL) trans-splicing, which involves an exon donated from a specialized SL RNA. Both depend primarily on the same signals and components as cis-splicing. Genic trans-splicing events producing protein-coding mRNAs have been described in a variety of organisms, including Caenorhabditis elegans and Drosophila. In mammalian cells, genic trans-splicing can be associated with cancers and translocations. SL trans-splicing has mainly been studied in nematodes and trypanosomes, but there are now numerous and diverse phyla (including primitive chordates) where this type of trans-splicing has been detected. Such diversity raises questions as to the evolutionary origin of the process. Another intriguing question concerns the function of trans-splicing, as operon resolution can only account for a small proportion of the total amount of SL trans-splicing. (C) 2011 John Wiley & Sons, Ltd. WIREs RNA 2011 2 417-434 DOI: 10.1002/wrna.71

In Caenorhabditis elegans, newly transcribed RNA is processed in several novel ways. Although introns are removed by a canonical spliceosome, they have evolved several specialized features that reflect the differences in the way they are recognized and the way they are spliced. C. elegans introns are unusually short, in part because they have no specific branch-point sequences and contain minimal polypryimidine tracts. Instead, their 3' splice site is characterized by a highly conserved consensus sequence, which alone may be sufficient to position all spliceosomal elements at the 3' end of the intron. Many RNA molecules are also trans-spliced: a capped 22 nt RNA leader is donated by one of a family of specialized snRNPs and spliced to an unpaired 3' splice site, usually just upstream of the start codon. The RNA upstream of this splice site, the outron, is removed during trans-splicing and presumably degraded, making the identification of the transcriptional start site problematic. Transcripts from approximately 70% of all genes are trans-spliced. Trans-splicing has enabled the evolution of operons - multigene clusters in which a single upstream promoter drives the transcription of a polycistronic pre-mRNA. The C. elegans genome contains more than 1000 such operons. The polycistronic pre-mRNA is processed into individual gene-encoding mRNAs by coordinated upstream 3' end formation and downstream trans-splicing. An intercistronic RNA sequence, the Ur element, plays a key role in specifying downstream trans-splicing.

INTRODUCTIONCaenorhabditis elegans is one of a number of organisms in which spliced leader (SL) trans-splicing contributes to the maturation of many pre-mRNA transcripts. C. elegans and related nematodes have two distinct classes of SL RNA—SL1 and SL2—that are used differentially for different classes of pre-mRNAs; namely, SL1 trans-splices non-operon genes and first genes in operons, while SL2 splices downstream operon genes. In vitro RNA splicing has previously been used to analyze substrate RNA sequences, required protein and RNA factors, and splicing mechanisms in a variety of systems. This protocol describes an in vitro splicing assay using C. elegans extract that promotes both cis- and trans-splicing of substrate RNAs. It has the ability to correctly specify and detect SL1 versus SL2 trans-splicing. Operon-derived substrates splice SL2, while substrates with an intron-like sequence followed by a 3′ trans-splice site (outron) splice SL1. Unlabeled T7-transcribed RNA substrates are incubated with crude embryonic extract and trans-spliced by endogenous SL small nuclear ribonucleoproteins (snRNPs). Spliced RNA is analyzed using a reverse transcriptase polymerase chain reaction (RT-PCR). Specific RT and PCR primers ensure detection of trans-spliced substrate RNA and allow a means to distinguish between SL1 and SL2 trans-spliced products. Substrates containing introns are also cis-spliced. All reaction products are ATP-dependent.

The heptad repeat of the RNA polymerase II (RNAPII) C-terminal domain is phosphorylated at serine 5 near gene 5' ends and serine 2 near 3' ends in order to recruit pre-mRNA processing factors. Ser-5(P) is associated with gene 5' ends to recruit capping enzymes, whereas Ser-2(P) is associated with gene 3' ends to recruit cleavage and polyadenylation factors. In the gene clusters called operons in Caenorhabditis elegans, there is generally only a single promoter, but each gene in the operon forms a 3' end by the usual mechanism. Although downstream operon genes have 5' ends, they receive their caps by trans splicing rather than by capping enzymes. Thus, they are predicted to not need Ser-5 phosphorylation. Here we show by RNAPII chromatin immunoprecipitation (ChIP) that internal operon gene 5' ends do indeed lack Ser-5(P) peaks. In contrast, Ser-2(P) peaks occur at each mRNA 3' end, where the 3'-end formation machinery binds. These results provide additional support for the idea that the serine phosphorylation of the C-terminal domain (CTD) serves to bring RNA-processing enzymes to the transcription complex. Furthermore, these results provide a novel demonstration that genes in operons are cotranscribed from a single upstream promoter.

Trans-splicing of one of two short leader RNAs, SL1 or SL2, occurs at the 5' 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 trans-spliced 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.

Spliced leader (SL) trans-splicing in Caenorhabditis elegans attaches a 22-nucleotide (nt) exon onto the 5' end of many mRNAs. A particular class of SL, SL2, splices mRNAs of downstream operon genes. Here we use an embryonic extract-based in vitro splicing system to show that SL2 specificity information is encoded within the polycistronic pre-mRNA, and that trans-splicing specificity is recapitulated in vitro. We define an RNA sequence required for SL2 trans-splicing, the U-rich (Ur) element, through mutational analysis and bioinformatics as a short stem-loop followed by a sequence motif, UAYYUU, located approximately 50 nt upstream of the trans-splice site. Furthermore, this element is predicted in intercistronic regions of numerous operons of C. elegans and other species that use SL2 trans-splicing. We propose that the UAYYUU motif hybridizes with the 5' splice site on the SL2 RNA to recruit the SL to the pre-mRNA. In this way, the UAYYUU motif in the pre-mRNA would serve an analogous function to the similar sequence in the U1 snRNA, which binds to the 5' splice site of introns, effectively reversing the roles of snRNP and pre-mRNA in trans-splicing.

In Caenorhabditis elegans, the transcripts of many genes are trans-spliced to an SL1 spliced leader, a process that removes the RNA extending from the transcription start site to the trans-splice site, thereby making it difficult to determine the position of the promoter. Here we use RT-PCR to identify promoters of trans-spliced genes. Many genes in C. elegans are organized in operons where genes are closely clustered, typically separated by only ~100 nucleotides, and transcribed by an upstream promoter. The transcripts of downstream genes are trans-spliced to an SL2 spliced leader. The polycistronic precursor RNA is processed into individual transcripts by 3' end formation and trans-splicing. Although the SL2 spliced leader does not appear to be used for other gene arrangements, there is a relatively small number of genes whose transcripts are processed by SL2 but are not close to another gene in the same orientation. Although these genes do not appear to be members of classical C. elegans operons, we investigated whether these might represent unusual operons with long spacing or a different, nonoperon mechanism for specifying SL2 trans-splicing. We show transcription of the entire region between the SL2 trans-spliced gene and the next upstream gene, sometimes several kilobases distant, suggesting that these represent exceptional operons. We also report a second type of atypical "alternative" operon, in which 3' end formation and trans-splicing by SL2 occur within an intron. In this case, the processing sometimes results in a single transcript, and sometimes in two separate mRNAs.