Short Exons Research Articles

Disruption of the EGFR E884–R958 ion pair conserved in the human kinome differentially alters signaling and inhibitor sensitivity

Targeted therapy against epidermal growth factor receptor (EGFR) represents a major therapeutic advance in lung cancer treatment. Somatic mutations of the EGFR gene, most commonly L858R (exon 21) and short in-frame exon 19 deletions, have been found to confer enhanced sensitivity towards the inhibitors gefitinib and erlotinib. We have recently identified an EGFR mutation E884K, in combination with L858R, in a patient with advanced lung cancer who progressed on erlotinib maintenance therapy, and subsequently had leptomeningeal metastases that responded to gefitinib. The somatic E884K substitution appears to be relatively infrequent, and resulted in a mutant lysine residue that disrupts an ion pair with residue R958 in the EGFR kinase domain C-lobe, an interaction that is highly conserved within the human kinome as demonstrated by our sequence analysis and structure analysis. Our studies here, using COS-7 transfection model system, show that E884K works in concert with L858R in-cis, in a dominant fashion, to change downstream signaling, differentially induce MAPK-ERK1/2 signaling and associated cell proliferation, and differentially alter sensitivity of EGFR phosphorylation inhibition by ERBB family inhibitors in an inhibitor-specific fashion. Mutations of the conserved ion pair E884-R958 may result in conformational changes that alter kinase substrate recognition. The analogous E1271K-MET mutation conferred differential sensitivity towards preclinical MET inhibitors SU11274 (unchanged), and PHA665752 (more sensitive). Systematic bioinformatics analysis of the mutation catalog in the human kinome (COSMIC) revealed the presence of cancer-associated mutations involving the conserved E884 homologous residue, and adjacent residues at the ion pair, in known proto-oncogenes (KIT, RET, MET, FAK) and tumor suppressor gene (LKB1). Targeted therapy using small molecule inhibitors should take into account potential cooperative effects of multiple kinase mutations, and their specific effects on downstream signaling and inhibitor sensitivity. Improved efficacy of targeted kinase inhibitors may be achieved by targeting the dominant activating mutations present.

Origination of the Split Structure of Spliceosomal Genes from Random Genetic Sequences

The mechanism by which protein-coding portions of eukaryotic genes came to be separated by long non-coding stretches of DNA, and the purpose for this perplexing arrangement, have remained unresolved fundamental biological problems for three decades. We report here a plausible solution to this problem based on analysis of open reading frame (ORF) length constraints in the genomes of nine diverse species. If primordial nucleic acid sequences were random in sequence, functional proteins that are innately long would not be encoded due to the frequent occurrence of stop codons. The best possible way that a long protein-coding sequence could have been derived was by evolving a split-structure from the random DNA (or RNA) sequence. Results of the systematic analyses of nine complete genome sequences presented here suggests that perhaps the major underlying structural features of split-genes have evolved due to the indigenous occurrence of split protein-coding genes in primordial random nucleotide sequence. The results also suggest that intron-rich genes containing short exons may have been the original form of genes intrinsically occurring in random DNA, and that intron-poor genes containing long exons were perhaps derived from the original intron-rich genes.

Pre-mRNA splicing: a complex picture in higher definition

Intron excision from pre-mRNAs of higher eukaryotes requires a transition from splice-site recognition across short exons to organization of the spliceosome across long introns. Recently, insight into this transition has been provided and, in addition, it has been shown that an alternative splicing factor, the polypyrimidine-tract-binding protein, can exert its control on splice-site choice by blocking this key step in the assembly of the splicing machinery.

The calcium channel β2 (CACNB2) subunit repertoire in teleosts

BackgroundCardiomyocyte contraction is initiated by influx of extracellular calcium through voltage-gated calcium channels. These oligomeric channels utilize auxiliary β subunits to chaperone the pore-forming α subunit to the plasma membrane, and to modulate channel electrophysiology [1]. Several β subunit family members are detected by RT-PCR in the embryonic heart. Null mutations in mouse β2, but not in the other three β family members, are embryonic lethal at E10.5 due to defects in cardiac contractility [2]. However, a drawback of the mouse model is that embryonic heart rhythm is difficult to study in live embryos due to their intra-uterine development. Moreover, phenotypes may be obscured by secondary effects of hypoxia. As a first step towards developing a model for contributions of β subunits to the onset of embryonic heart rhythm, we characterized the structure and expression of β2 subunits in zebrafish and other teleosts.ResultsCloning of two zebrafish β2 subunit genes (β2.1 and β2.2) indicated they are membrane-associated guanylate kinase (MAGUK)-family genes. Zebrafish β2 genes show high conservation with mammals within the SH3 and guanylate kinase domains that comprise the "core" of MAGUK proteins, but β2.2 is much more divergent in sequence than β2.1. Alternative splicing occurs at the N-terminus and within the internal HOOK domain. In both β2 genes, alternative short ATG-containing first exons are separated by some of the largest introns in the genome, suggesting that individual transcript variants could be subject to independent cis-regulatory control. In the Tetraodon nigrovidis and Fugu rubripes genomes, we identified single β2 subunit gene loci. Comparative analysis of the teleost and human β2 loci indicates that the short 5' exon sequences are highly conserved. A subset of 5' exons appear to be unique to teleost genomes, while others are shared with mammals. Alternative splicing is temporally and spatially regulated in embryo and adult. Moreover, a different subset of spliced β2 transcript variants is detected in the embryonic heart compared to the adult.ConclusionThese studies refine our understanding of β2 subunit diversity arising from alternative splicing, and provide the groundwork for functional analysis of β2 subunit diversity in the embryonic heart.

In vivo translation and stability of trans-spliced mRNAs in nematode embryos

Spliced leader trans-splicing adds a short exon, the spliced leader (SL), to pre-mRNAs to generate 5′ ends of mRNAs. Addition of the SL in metazoa also adds a new cap to the mRNA, a trimethylguanosine (m 3 2,2,7GpppN) (TMG) that replaces the typical eukaryotic monomethylguanosine (m 7GpppN)(m 7G) cap. Both trans-spliced (m 3 2,2,7GpppN-SL-RNA) and not trans-spliced (m 7GpppN-RNA) mRNAs are present in the same cells. Previous studies using cell-free systems to compare the overall translation of trans-spliced versus non-trans-spliced RNAs led to different conclusions. Here, we examine the contribution of m 3 2,2,7GpppG-cap and SL sequence and other RNA elements to in vivo mRNA translation and stability in nematode embryos. Although 70–90% of all nematode mRNAs have a TMG-cap, the TMG cap does not support translation as well as an m 7G-cap. However, when the TMG cap and SL are present together, they synergistically interact and translation is enhanced, indicating both trans-spliced elements are necessary to promote efficient translation. The SL by itself does not act as a cap-independent enhancer of translation. The poly(A)-tail synergistically interacts with the mRNA cap enhancing translation and plays a greater role in facilitating translation of TMG-SL mRNAs. In general, recipient mRNA sequences between the SL and AUG and the 3′ UTR do not significantly contribute to the translation of trans-spliced mRNAs. Overall, the combination of TMG cap and SL contribute to mRNA translation and stability in a manner typical of a eukaryotic m 7G-cap and 5′ UTRs, but they do not differentially enhance mRNA translation or stability compared to RNAs without the trans-spliced elements.

Histone H1‐like, lysine‐rich low complexity amino acid extensions in mosquito ribosomal proteins RpL23a and RpS6 have evolved independently

Histone H1-like amino acid extensions have been described at the amino terminus of Drosophila RpL22 and RpL23a, and at the carboxyl terminus of mosquito ribosomal protein RpS6. An in silico search suggested that RpL23a, but not RpL22, in Anopheles gambiae has an amino-terminal extension. Because low complexity amino acid extensions are not common on eukaryotic ribosomal proteins, and their functions are unknown, we cloned cDNAs encoding RpL23a from Aedes albopictus and Anopheles stephensi mosquito cell lines. RpL23a proteins in Aedes and Anopheles mosquitoes are rich in lysine (approximately 25%), alanine (approximately 21%), and proline (approximately 8%), have a mass of approximately 40 kDa, a pI of 11.4 to 11.5, and contain an N-terminal extension of approximately 260 amino acid residues. The N-terminal extension in mosquito RpL23a is about 100 amino acids longer than that in the Drosophila RpL23a homolog, and contains several repeated amino acid motifs. Analysis of exon-intron organization in the An. gambiae and in D. melanogaster genes suggests that a short first exon encodes a series of 11 amino acid residues conserved in RpL23a proteins from Drosophila, mosquitoes, and the moth, Bombyx mori. The histone H1-like sequence in RpL23a is encoded entirely within the second exon. The C-terminal 126 amino acid residues of the RpL23a protein, encoded by exon 3 in Drosophila, and by exons 3 and 4 in Anopheles gambiae, are well conserved, and correspond to Escherichia coli RpL23 with the addition of the eukaryotic N-terminal nuclear localization sequence. Sequence comparisons indicate that the histone H1-like extensions on mosquito RpS6 and RpL23a have evolved independently of each other, and of histone H1 proteins.

In search of the small ones: improved prediction of short exons in vertebrates, plants, fungi and protists

Prediction of the coding potential for stretches of DNA is crucial in gene calling and genome annotation, where it is used to identify potential exons and to position their boundaries in conjunction with functional sites, such as splice sites and translation initiation sites. The ability to discriminate between coding and non-coding sequences relates to the structure of coding sequences, which are organized in codons, and by their biased usage. For statistical reasons, the longer the sequences, the easier it is to detect this codon bias. However, in many eukaryotic genomes, where genes harbour many introns, both introns and exons might be small and hard to distinguish based on coding potential. Here, we present novel approaches that specifically aim at a better detection of coding potential in short sequences. The methods use complementary sequence features, combined with identification of which features are relevant in discriminating between coding and non-coding sequences. These newly developed methods are evaluated on different species, representative of four major eukaryotic kingdoms, and extensively compared to state-of-the-art Markov models, which are often used for predicting coding potential. The main conclusions drawn from our analyses are that (1) combining complementary sequence features clearly outperforms current Markov models for coding potential prediction in short sequence fragments, (2) coding potential prediction benefits from length-specific models, and these models are not necessarily the same for different sequence lengths and (3) comparing the results across several species indicates that, although our combined method consistently performs extremely well, there are important differences across genomes. http://bioinformatics.psb.ugent.be/.

Mutations in the SF1-U2AF59-U2AF23 Complex Cause Exon Skipping in Schizosaccharomyces pombe

To identify genes involved in the mechanism to ensure ordered 5' to 3' exon joining in constitutively spliced pre-mRNAs, we screened for mutants that cause exon skipping in the fission yeast Schizosaccharomyces pombe using a reporter plasmid, which contains the ura4+ gene with the nda3 intron 1-exon 2-intron 2 sequence. The reporter plasmid was designed to produce the functional ura4+ mRNA, when the central nda3 exon is skipped during the splicing reaction. We mutagenized cells harboring the plasmid by UV irradiation and isolated 34 ura+ mutants that grew on minimal medium. Of those, eight mutants were found to be temperature sensitive (ts) for growth. Complementation analyses revealed that the ts mutants belong to three distinct complementation groups named ods (ordered splicing) 1, 2, and 3. RT-PCR analyses showed that products of exon skipping were actually generated in the ods mutants. We cloned the genes responsible for the ods mutations, and found that ods1+, ods2+, and ods3+ encode splicing factors Prp2p/U2AF59, U2AF23, and SF1, respectively, which form a SF1-U2AF59-U2AF23 complex involved in recognition of the branch-point and 3' splice site sequences in a pre-mRNA. We also showed that mutations in the SF1-U2AF59-U2AF23 binding sequences in the reporter plasmid result in exon skipping in wild-type S. pombe cells. In addition, drugs that decrease the rate of transcription elongation were found to suppress the exon skipping in the ods mutants. These results suggest that co-transcriptional recognition of a nascent pre-mRNA by the SF1-U2AF59-U2AF23 complex is essential for ordered exon joining in constitutive splicing in S. pombe.

Targeted Skipping of a Single Exon Harboring a Premature Termination Codon Mutation: Implications and Potential for Gene Correction Therapy for Selective Dystrophic Epidermolysis Bullosa Patients

This study examined the feasibility of antisense oligoribonucleotide (AON) therapy for dystrophic epidermolysis bullosa (DEB). AON was designed to induce skipping of a targeted exon containing a premature termination codon mutation, resulting in restoration of the open reading frame. We targeted exon 70 of COL7A1, as a recurrent mutation 5818delC in Japanese DEB patients was localized to exon 70. We found that one AON induced effective skipping of normal exon 70 containing 16 amino acids. Attachment and migration analyses showed that recombinant collagen without contribution of exon 70 was similar in effect to normal type VII collagen. Next, we synthesized mutation-specific AON by deleting cytosine at 5818. Introduction of this AON into DEB keratinocytes harboring 5818delC showed that the AON induced skipping of exon 70 in the abnormal 5818delC allele. Furthermore, 6.2% of DEB keratinocytes started to express type VII collagen in vitro after application of the mutation-specific AON. Injection of the AON into rat model grafted with DEB keratinocytes and fibroblasts induced a low amount of type VII collagen expression. We conclude that skipping of targeted exons using mutation-specific AON may show potential for future gene therapy for DEB patients.

A Genome-Wide Analysis Indicates that Yeast Pre-mRNA Splicing Is Predominantly Posttranscriptional

Recent ChIP experiments indicate that spliceosome assembly and splicing can occur cotranscriptionally in S. cerevisiae. However, only a few genes have been examined, and all have long second exons. To extend these studies, we analyzed intron-containing genes with different second exon lengths by using ChIP as well as whole-genome tiling arrays (ChIP-CHIP). The data indicate that U1 snRNP recruitment is independent of exon length. Recursive splicing constructs, which uncouple U1 recruitment from transcription, suggest that cotranscriptional U1 recruitment contributes to optimal splicing efficiency. In contrast, U2 snRNP recruitment, as well as cotranscriptional splicing, is deficient on short second exon genes. We estimate that > or =90% of endogenous yeast splicing is posttranscriptional, consistent with an analysis of posttranscriptional snRNP-associated pre-mRNA.

The strength of the HIV-1 3' splice sites affects Rev function

BackgroundThe HIV-1 Rev protein is a key component in the early to late switch in HIV-1 splicing from early intronless (e.g. tat, rev) to late intron-containing Rev-dependent (e.g. gag, vif, env) transcripts. Previous results suggested that cis-acting sequences and inefficient 5' and 3' splice sites are a prerequisite for Rev function. However, we and other groups have shown that two of the HIV-1 5' splice sites, D1 and D4, are efficiently used in vitro and in vivo. Here, we focus on the efficiency of the HIV-1 3' splice sites taking into consideration to what extent their intrinsic efficiencies are modulated by their downstream cis-acting exonic sequences. Furthermore, we delineate their role in RNA stabilization and Rev function.ResultsIn the presence of an efficient upstream 5' splice site the integrity of the 3' splice site is not essential for Rev function whereas an efficient 3' splice site impairs Rev function. The detrimental effect of a strong 3' splice site on the amount of Rev-dependent intron-containing HIV-1 glycoprotein coding (env) mRNA is not compensatable by weakening the strength of the upstream 5' splice site. Swapping the HIV-1 3' splice sites in an RRE-containing minigene, we found a 3' splice site usage which was variably dependent on the presence of the usual downstream exonic sequence. The most evident activation of 3' splice site usage by its usual downstream exonic sequence was observed for 3' splice site A1 which was turned from an intrinsic very weak 3' splice site into the most active 3' splice site, even abolishing Rev activity. Performing pull-down experiments with nuclear extracts of HeLa cells we identified a novel ASF/SF2-dependent exonic splicing enhancer (ESE) within HIV-1 exon 2 consisting of a heptameric sequence motif occurring twice (M1 and M2) within this short non-coding leader exon. Single point mutation of M1 within an infectious molecular clone is detrimental for HIV-1 exon 2 recognition without affecting Rev-dependent vif expression.ConclusionUnder the conditions of our assay, the rate limiting step of retroviral splicing, competing with Rev function, seems to be exclusively determined by the functional strength of the 3' splice site. The bipartite ASF/SF2-dependent ESE within HIV-1 exon 2 supports cross-talk between splice site pairs across exon 2 (exon definition) which is incompatible with processing of the intron-containing vif mRNA. We propose that Rev mediates a switch from exon to intron definition necessary for the expression of all intron-containing mRNAs.

Vascular-Specific Increase in Exon 1b-Encoded Ca V1.2 Channels in Spontaneously Hypertensive Rats

The vasculature of the adult spontaneously hypertensive rat (SHR) is known to express more functional L-type Ca channels than the vasculature of normotensive Wistar Kyoto (WKY) rats, but it is not known which Ca(V)1.2 channel isoform is upregulated. Western blots and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) were used to compare the expression levels of Ca(V)1.2 channel protein and message in selected tissues of adult SHR and WKY rats. The results indicate overexpression in SHR vasculature specifically of the short exon 1b-encoded amino terminus Ca(V)1.2 isoform. Brain and visceral smooth muscle expressing the same isoform were not similarly affected. Differences in message levels are insufficient to account for the differences in isoform-specific protein levels. We conclude that SHR vasculature must regulate the channel postranscriptionally. Further experiments will be required to determine whether this involves translation of protein from exon 1b-specific transcripts more efficiently, posttranslational chaperoning to the surface membrane more efficiently, or selective degradation of the short amino terminus form of the protein more slowly than in WKY vasculature.

Origin and evolution of the TKDP gene family

The placenta is arguably the most diverse mammalian organ, and several gene families expressed in trophoblast tissue are evolving unusually rapidly. The trophoblast Kunitz domain protein ( TKDP) gene family arose relatively recently within the Artiodactyla order in the lineage leading to modern day ruminants. Each contains a classical Kunitz-type serine peptidase inhibitory domain at the carboxyl-terminus and one or more unique N-domains preceded by the signal peptide/propeptide (SP/PP) region. We isolated, mapped and fully sequenced the ovine (ov) TKDP-1 gene. Making use of available cDNA sequences to other TKDP family members and recent information from the bovine genome sequence, we have been able to make inferences about the origin and evolution of the entire gene family. The crucial event that created the initial TKDP gene was when a short exon was inserted into the intron that separated the sequences encoding the SP/PP and Kunitz domains of an ancestral Kunitz gene. This short exon was flanked by endogenous retroviral repetitive elements that likely promoted subsequent duplication events to provide a tripartite N-domain containing three repeats. This three-exon structure further duplicated once in its entirety to provide the progenitor of the present day ovTKDP-1 gene, which has two N-domains, and most probably twice to provide the progenitor of the bovine TKDP-1 gene, which has three N-domains. The TKDPs provide an example of a gene family that is evolving rapidly by whole gene duplication, intragenic duplication events, and selection for amino acid (aa) change.

Challenging the spliceosome machine

Analysis of a set of almost 25,000 donor and acceptor splice sites in Drosophila shows that information content increases near splice sites flanking very long of very short introns and exons.

Intragenic Hill-Robertson Interference Influences Selection Intensity on Synonymous Mutations in Drosophila

Natural selection influences synonymous mutations and synonymous codon usage in many eukaryotes to improve the efficiency of translation in highly expressed genes. Recent studies of gene composition in eukaryotes have shown that codon usage also varies independently of expression levels, both among genes and at the intragenic level. Here, we investigate rates of evolution (Ks) and intensity of selection (gamma(s)) on synonymous mutations in two groups of genes that differ greatly in the length of their exons, but with equivalent levels of gene expression and rates of crossing-over in Drosophila melanogaster. We estimate gamma(s) using patterns of divergence and polymorphism in 50 Drosophila genes (100 kb of coding sequence) to take into account possible variation in mutation trends across the genome, among genes or among codons. We show that genes with long exons exhibit higher Ks and reduced gamma(s) compared to genes with short exons. We also show that Ks and gamma(s) vary significantly across long exons, with higher Ks and reduced gamma(s) in the central region compared to flanking regions of the same exons, hence indicating that the difference between genes with short and long exons can be mostly attributed to the central region of these long exons. Although amino acid composition can also play a significant role when estimating Ks and gamma(s), our analyses show that the differences in Ks and gamma(s) between genes with short and long exons and across long exons cannot be explained by differences in protein composition. All these results are consistent with the Interference Selection (IS) model that proposes that the Hill-Robertson (HR) effect caused by many weakly selected mutations has detectable evolutionary consequences at the intragenic level in genomes with recombination. Under the IS model, exon size and exon-intron structure influence the effectiveness of selection, with long exons showing reduced effectiveness of selection when compared to small exons and the central region of long exons showing reduced intensity of selection compared to flanking coding regions. Finally, our results further stress the need to consider selection on synonymous mutations and its variation--among and across genes and exons--in studies of protein evolution.

The effect of U1 snRNA binding free energy on the selection of 5′ splice sites

The importance of U1 snRNA binding free energy in the regulation of alternative splicing has been studied in some genes with site-directed mutagenesis. Here we report a large-scale analysis of its impact on 5′ splice site (5′ss) selection in human genome. The results show that free energy exerts different effects on alternative 5′ss choice in different situations and −8.1 kcal/mol is a threshold. When both free energies of two competing 5′ss are larger than −8.1 kcal/mol, the 5′ss with lower free energy is more frequently used. However, in other pairs of 5′ss, lower-free-energy 5′ss does not seem to be favored and even the other 5′ss is used more frequently, which suggests that very low binding free energy would impair splicing. Some observations hold true only for those alternative 5′ splicing with short alternative exons (<50nt), which implies a complex mechanism of 5′ss selection involving both U1 snRNA binding free energy and regulatory factors.

Effective statistical features for coding and non-coding DNA sequence classification for yeast, C. elegans and human

This study performs a quantitative evaluation of the different coding features in terms of their information content for the classification of coding and non-coding regions for three species. Our study indicated that coding features that are effective for yeast or C. elegans are generally not very effective for human, which has a short average exon length. By performing a correlation analysis, we identified a subset of human coding features with high discriminative power, but complementary in their information content. For this subset, a classification accuracy of up to 90% was obtained using a simple kNN classifier.

Identification of the functional elements in the promoter region of human DNA topoisomerase IIIβ gene

In this study, we have isolated and characterized the promoter region of the human DNA topoisomerase IIIβ ( hTOP3β) gene. The 5′ RACE assay showed a short exon 1 encoding only the 35-bp untranslated region and suggested the presence of multiple transcription initiation sites. The hTOP3β gene promoter lacks a canonical TATA box or initiation element and is moderately high in GC content. Transient expression of a luciferase reporter gene under the control of serially deleted 5′-flanking sequence identified an activator element between −141 and −119 upstream of the transcription initiation site and a second regulatory element between −91 and −71. On the basis of scanning mutations of triple nucleotides, we demonstrated that a 5′GGAACC3′ element between −117 and −112 plays a critical role in the up-regulation of the basal transcription activity. Changing the 5′GGAACC3′ sequence leads to markedly reduced promoter activity. Gel mobility shift assays revealed that the 5′GGAACC3′ element is required for DNA binding by the transcription factor complex. These observations lead to the conclusion that the positive regulatory region including the 5′GGAACC3′ core element is essential for efficient expression of the hTOP3β gene as well as for the binding of as yet unidentified regulatory factor(s).

Identification of promoter region of ghrelin gene in human medullary thyroid carcinoma cell line

Ghrelin, an endogenous ligand for growth hormone (GH) secretagogue receptor, stimulates GH secretion. The ghrelin gene is expressed most abundantly in stomach. The mRNA is also detected in other tissues and cell lines. However, the mechanism of the transcriptional regulation of the ghrelin gene has not yet been clarified. In the present study, we have investigated the regulatory region of the ghrelin gene expression in the human medullary thyroid carcinoma cell line (TT cells). PCR analysis of the 5'-region for human ghrelin gene revealed the presence of the first exon corresponding to the short non-coding first exon of the mouse ghrelin gene. The first exon is located at the 502 bp upstream from the 5'-end of the formerly reported human ghrelin gene. RT-PCR analysis showed the expression of the first exon in the stomach and TT cells. The expression of the first exon in the human stomach was confirmed by 5'-RACE method. Significant level of promoter activity was observed in the 1225-1107 bp up-stream region of the translation initiation site by luciferase assay. Specific protein binding to the promoter region of -1129 to -1100 was detected by electrophoretic mobility shift assay with nuclear extract from TT cells. These results suggest that the ghrelin gene expression in TT cells might be regulated by the upstream region of the first exon.

Study of the mouse sortilin gene: Effects of its transient silencing by RNA interference in TM4 Sertoli cells.

Using databases of the mouse genome in combination with a sequence deduced from a mouse sortilin cDNA originated in our laboratory, we found the sortilin gene to map to a region of chromosome 3. The mouse sortilin gene contains 19 short exons separated by introns of various sizes. The study elucidated the exon-intron boundaries. Some introns extend over more than 24 kb. In the cytoplasmic domain of the translation product, we found a dileucine motif and three other motifs known to constitute the active sorting signal of the mannose 6-phosphate receptor (M6P-R). We also tested the hypothesis that sortilin is involved in the sorting of prosaposin (SGP-1) to the lysosomes. Prosaposin was initially identified in Sertoli cells, found in large amounts in the lysosomal compartment and implicated in the degradation of residual bodies released by the spermatids during spermiation. Interestingly, the targeting of prosaposin to the lysosomes is independent of the M6P-R. This investigation demonstrated that sortilin was required for the trafficking of prosaposin to the lysosomes in TM4 cells. The requirement of sortilin was shown using a siRNA probe to block the translation of sortilin mRNA. Sortilin-deficient cells were not able to route prosaposin to the lysosomal compartment but continue to transport cathepsin B, since this hydrolase uses the M6P-R to be routed to the lysosomes. These results indicate that sortilin appears to be involved in the lysosomal trafficking of prosaposin.