Effects of cocaine and withdrawal on the mouse nucleus accumbens transcriptome.

Genetic association studies, pharmacological investigations and analysis of mice-lacking individual genes have made it clear that Cocaine administration and Withdrawal have a profound impact on multiple neurotransmitter systems. The GABAergic medium spiny neurons of the nucleus accumbens (NAc) exhibit changes in the expression of genes encoding receptors for glutamate and in the signaling pathways triggered by dopamine binding to G-protein-coupled dopamine receptors. Deep sequence analysis provides a sensitive, quantitative and global analysis of the effects of Cocaine on the NAc transcriptome. RNA prepared from the NAc of adult male mice receiving daily injections of Saline or Cocaine, or Cocaine followed by a period of Withdrawal, was used for high-throughput sequence analysis. Changes were validated by quantitative polymerase chain reaction or Western blot. On the basis of pathway analysis, a preponderance of the genes affected by Cocaine and Withdrawal was involved in the cadherin, heterotrimeric G-protein and Wnt signaling pathways. Distinct subsets of cadherins and protocadherins exhibited a sustained increase or decrease in expression. Sustained down-regulation of several heterotrimeric G-protein ß- and γ-subunits was observed. In addition to altered expression of receptors for small molecule neurotransmitters, neuropeptides and endocannabinoids, changes in the expression of plasma membrane transporters and vesicular neurotransmitter transporters were also observed. The effects of chronic Cocaine and Withdrawal on the expression of genes essential to cholinergic, glutamatergic, GABAergic, peptidergic and endocannabinoid signaling are as profound as their effects on dopaminergic transmission. Simultaneous targeting of multiple Withdrawal-specific changes in gene expression may facilitate development of new therapeutic approaches that are better able to prevent relapse.

Alternative splicing of the Drosophila Dscam pre-mRNA is both temporally and spatially regulated.

The Drosophila melanogaster Down syndrome cell adhesion molecule (Dscam) gene encodes an axon guidance receptor that can express 38,016 different mRNAs by virtue of alternative splicing. The Dscam gene contains 95 alternative exons that are organized into four clusters of 12, 48, 33, and 2 exons each. Although numerous Dscam mRNA isoforms can be synthesized, it remains to be determined whether different Dscam isoforms are synthesized at different times in development or in different tissues. We have investigated the alternative splicing of the Dscam exon 4 cluster, which contains 12 mutually exclusive alternative exons, and found that Dscam exon 4 alternative splicing is developmentally regulated. The most highly regulated exon, 4.2, is infrequently used in early embryos but is the predominant exon 4 variant used in adults. Moreover, the developmental regulation of exon 4.2 alternative splicing is conserved in D. yakuba. In addition, different adult tissues express distinct collections of Dscam mRNA isoforms. Given the role of Dscam in neural development, these results suggest that the regulation of alternative splicing plays an important role in determining the specificity of neuronal wiring. In addition, this work provides a framework to determine the mechanisms by which complex alternative splicing events are regulated.

The role of U2AF35 and U2AF65 in enhancer-dependent splicing.

Splicing enhancers are RNA sequence elements that promote the splicing of nearby introns. The mechanism by which these elements act is still unclear. Some experiments support a model in which serine-arginine (SR)-rich proteins function as splicing activators by binding to enhancers and recruiting the splicing factor U2AF to an adjacent weak 3' splice site. In this model, recruitment requires interactions between the SR proteins and the 35-kDa subunit of U2AF (U2AF35). However, more recent experiments have not supported the U2AF recruitment model. Here we provide additional evidence for the recruitment model. First, we confirm that base substitutions that convert weak 3' splice sites to a consensus sequence, and therefore increase U2AF binding, relieve the requirement for a splicing activator. Second, we confirm that splicing activators are required for the formation of early spliceosomal complexes on substrates containing weak 3' splice sites. Most importantly, we find that splicing activators promote the binding of both U2AF65 and U2AF35 to weak 3' splice sites under splicing conditions. Finally, we show that U2AF35 is required for maximum levels of activator-dependent splicing. We conclude that a critical function of splicing activators is to recruit U2AF to the weak 3' splice sites of enhancer-dependent introns, and that efficient enhancer-dependent splicing requires U2AF35.

Alternative splicing: increasing diversity in the proteomic world.

How can the genome of Drosophila melanogaster contain fewer genes than the undoubtedly simpler organism Caenorhabditis elegans? The answer must lie within their proteomes. It is becoming clear that alternative splicing has an extremely important role in expanding protein diversity and might therefore partially underlie the apparent discrepancy between gene number and organismal complexity. Alternative splicing can generate more transcripts from a single gene than the number of genes in an entire genome. However, for the vast majority of alternative splicing events, the functional significance is unknown. Developing a full catalog of alternatively spliced transcripts and determining each of their functions will be a major challenge of the upcoming proteomic era.

A systematic analysis of the factors that determine the strength of pre-mRNA splicing enhancers.

We find that the strength of splicing enhancers is determined by the relative activities of the bound serine-arginine (SR)-rich splicing factors, the number of SR proteins within the enhancer complex and the distance between the enhancer and the intron. Remarkably, the splicing activity of the bound SR proteins is directly proportional to the number of RS tetrapeptide sequences within the RS domain. Quantitative analysis of the effects of varying the distance between the enhancer and the intron revealed that the splicing efficiency is directly proportional to the calculated probability of a direct interaction between the enhancer complex and the 3' splice site. These data are consistent with a model in which splicing enhancers function by increasing the local concentration of SR proteins in the vicinity of the nearby intron through RNA looping.

Arginine/serine-rich domains of SR proteins can function as activators of pre-mRNA splicing.

Serine/arginine (SR)-rich splicing factors contain an RNA binding domain and an arginine/serine (RS)-rich domain required for protein-protein interactions. In addition to their roles in the basic splicing reaction, SR proteins function as components of splicing enhancer complexes. Here, we investigate the role of RS domains in splicing enhancer function. Hybrid proteins containing RS domains fused to the MS2 RNA binding protein were tested in vitro with RNA substrates bearing an MS2 recognition sequence. These hybrid proteins activated splicing in nuclear extracts, but not in S100 extracts lacking SR proteins. However, intact recombinant SR proteins could complement the activity of the hybrid proteins in S100 extracts. These data demonstrate that RS domains function as splicing activators and suggest that the general and enhancer-dependent functions of SR proteins can be uncoupled.

Restoration of both structure and function to a defective poly(A) site by in vitro selection.

Efficient cleavage and polyadenylation at the human immunodeficiency virus type-1 (HIV-1) poly(A) site requires an upstream 3'-processing enhancer to overcome the suboptimal sequence context of the AAUAAA hexamer. The HIV-1 3'-processing enhancer functions to stabilize the association of the pre-mRNA with cleavage and polyadenylation specificity factor (CPSF), the factor responsible for recognition of the AAUAAA hexamer. Intriguingly, in the absence of the 3'-processing enhancer, CPSF binding and polyadenylation efficiency could be restored to near wild-type levels upon replacement of the 14-nucleotide region immediately 5' of the HIV-1 AAUAAA hexamer (the B segment) by the analogous sequences from the efficient adenovirus L3 poly(A) site. To further investigate the contributions of RNA sequence and structure to poly(A) site recognition, we have used an in vitro selection system to identify B segment sequences that enhance the polyadenylation efficiency of a pre-cleaved RNA lacking a 3'-processing enhancer. The final RNA selection pool was composed of two predominant classes of RNAs. Nuclease probing revealed that the selected sequences restored an RNA conformation that facilitates recognition of the AAUAAA hexamer by CPSF. These results indicate that both the sequence and structural context of the AAUAAA hexamer contribute to poly(A) site recognition by CPSF.

TFIIS binds to mouse RNA polymerase I and stimulates transcript elongation and hydrolytic cleavage of nascent rRNA.

Efficient transcription elongation by RNA polymerase I (Pol I) requires a specific Pol I-associated factor, termed TIF-IC. Here we show that TFIIS, a factor that has previously been shown to promote read-through past many types of blocks to elongation by RNA polymerase II, also enhances Pol I-directed transcription elongation. In a reconstituted transcription system containing purified proteins, TFIIS stimulates Pol I transcription by increasing the overall rate of RNA chain elongation. As with Pol II, ternary Pol I complexes cleave the 3' end of the nascent transcripts in response to TFIIS. The truncated RNAs remain bound to the template, are subject to pyrophosphorolysis, and can be chased into longer transcripts. Moreover, we show by immunoprecipitation and specific affinity chromatography that TFIIS physically interacts with Pol I. The results suggest that nascent transcript cleavage by TFIIS or a TFIIS-related factor may be a general mechanism by which both Pol I and Pol II can bypass transcriptional impediments.

RNA structure is a critical determinant of poly(A) site recognition by cleavage and polyadenylation specificity factor.

Sequence conservation among mammalian poly(A) sites is limited to the sequence AAUAAA, coupled with an amorphous downstream U- or GU-rich region. Since these sequences may also occur within the coding region of mRNAs, additional information must be required to define authentic poly(A) sites. Several poly(A) sites have been shown to contain sequences outside the core elements that enhance the efficiency of 3' processing in vivo and in vitro. The human immunodeficiency virus type 1, equine infectious anemia virus, and adenovirus L1 3' processing enhancers have been shown to promote the binding of cleavage and polyadenylation specificity factor (CPSF), the factor responsible for recognition of AAUAAA, to the pre-mRNA, thereby facilitating the assembly of a stable 3' processing complex. We have used in vitro selection to examine the mechanism by which the human immunodeficiency virus type 1 3' processing enhancer promotes the interaction of CPSF with the AAUAAA hexamer. Surprisingly, RNAs selected for efficient polyadenylation were related by structure rather than sequence. Therefore, in the absence of extensive sequence conservation, our results strongly suggest that RNA structure is a critical determinant of poly(A) site recognition by CPSF and may play a key role in poly(A) site definition.

A common mechanism for the enhancement of mRNA 3' processing by U3 sequences in two distantly related lentiviruses.

The protein coding regions of all retroviral pre-mRNAs are flanked by a direct repeat of R-U5 sequences. In many retroviruses, the R-U5 repeat contains a complete core poly(A) site-composed of a highly conserved AAUAAA hexamer and a GU-rich downstream element. A mechanism that allows for the bypass of the 5' core poly(A) site and the exclusive use of the 3' core poly(A) site must therefore exist. In human immunodeficiency virus type 1 (HIV-1), sequences within the U3 region appear to play a key role in poly(A) site selection. U3 sequences are required for efficient 3' processing at the HIV-1 poly(A) site both in vivo and in vitro. These sequences serve to promote the interaction of cleavage and polyadenylation specificity factor (CPSF) with the core poly(A) site. We have now demonstrated the presence of a functionally analogous 3' processing enhancer within the U3 region of a distantly related lentivirus, equine infectious anemia virus (EIAV). U3 sequences enhanced the processing of the EIAV core poly(A) site sevenfold in vitro. The U3 sequences also enhanced the stability of CPSF binding at the core poly(A) site. Optimal processing required the TAR RNA secondary structure that resides within the R region 28 nucleotides upstream of the AAUAAA hexamer. Disruption of TAR reduced processing, while compensatory changes that restored the RNA structure also restored processing to the wild-type level, suggesting a position dependence of the U3-encoded enhancer sequences. Finally, the reciprocal exchange of the EIAV and HIV U3 regions demonstrated the ability of each of these sequences to enhance both 3' processing and the binding of CPSF in the context of the heterologous core poly(A) site. The impact of U3 sequences upon the interaction of CPSF at the core poly(A) site may therefore represent a common strategy for retroviral poly(A) site selection.

CPSF recognition of an HIV-1 mRNA 3'-processing enhancer: multiple sequence contacts involved in poly(A) site definition.

The endonucleolytic cleavage and polyadenylation of a pre-mRNA in mammalian cells requires two cis-acting elements, a highly conserved AAUAAA hexamer and an amorphous U- or GU-rich downstream element, that together constitute the "core" poly(A) site. The terminal redundancy of the HIV-1 pre-mRNA requires that the processing machinery disregard a core poly(A) site at the 5' end of the transcript, and efficiently utilize an identical signal that resides near the 3' end. Efficient processing at the 3' core poly(A) site, both in vivo and in vitro, has been shown to require sequences 76 nucleotides upstream of the AAUAAA hexamer. In this report we demonstrate that this HIV-1 upstream element interacts directly with the 160-kD subunit of CPSF (cleavage polyadenylation specificity factor), the factor responsible for the recognition of the AAUAAA hexamer. The presence of the upstream element in the context of the AAUAAA hexamer directs the stable binding of CPSF to the pre-mRNA and enhances the efficiency of poly(A) addition in reactions reconstituted with purified CPSF and recombinant poly(A) polymerase. Our results indicate that the dependence of HIV-1 3' processing on upstream sequences is a consequence of the suboptimal sequence context of the AAUAAA hexamer. We suggest that poly(A) site definition involves the recognition of multiple heterogeneous sequence elements in the context of the AAUAAA hexamer.