transferase activities are part of the same polypeptide University of Oxford at the N terminus and C terminus, respectively, while South Parks Road in yeast they are catalyzed by separate enzymes. This Oxford OX1 3RE initial cap structure is then recognized by the cap bind-United Kingdom ing complex (CBC), which contains two proteins, CBP20 and CBP80. Upon export through the nuclear pore complex, the nuclear cap binding proteins are replaced by The messenger RNA processing reactions of capping, the cytoplasmic translation initiation factor, eIF-4E splicing, and polyadenylation occur cotranscription-(Shatkin and Manley, 2000, for review). ally. They not only influence one another's efficiency The cap structure bound to CBC is believed to play and specificity, but are also coordinated by transcripa major role in the stabilization of the mRNA, since it tion. The phosphorylated CTD of RNA polymerase II represents an obstacle for 5Ј-3Ј exonucleases (Beelman provides key molecular contacts with these mRNA and Parker, 1995). In addition, in the cytoplasm, cap processing reactions throughout transcriptional elonbound to eIF-4E and other translation initiation factors gation and termination.enhances translation by promoting the engagement of the ribosomal subunits with the mRNA. This is at least partly achieved by the interaction of eIF4G with poly(A)-

Reconstruction of a gene with its introns removed results in reduced levels of cytoplasmic mRNA. This is partly explained by introns promoting the export of mRNA through coupling splicing to nuclear export processes. However, we show here that splicing signals can have a direct role in enhancing gene transcription. Removal of promoter proximal splice signals from a mammalian gene or the excision of introns from two different yeast genes results in a marked reduction in levels of nascent transcription, based on both nuclear run-on and direct image analysis. This further establishes that mRNA processing and transcription are tightly coupled mechanisms.

Diverse classes of noncoding RNA, including small nuclear RNAs (snRNAs), play fundamental regulatory roles at many stages of gene expression. For example, recent studies have implicated 7SK RNA and components of the splicing apparatus in the regulation of transcriptional elongation. Here we present the first evidence of the involvement of an snRNA in the regulation of transcriptional initiation. We demonstrate that TFIIH, a general transcription initiation factor, specifically associates with U1 snRNA, a core-splicing component. Analysis of the TFIIH-dependent stages of transcription in a reconstituted system demonstrates that U1 stimulates the rate of formation of the first phosphodiester bond by RNA polymerase II. In addition, a promoter-proximal 5' splice site recognized by U1 snRNA stimulates TFIIH-dependent reinitiation of productive transcription. Our results suggest that U1 snRNA functions in regulating transcription by RNA Polymerase II in addition to its role in RNA processing.

African trypanosomes are not passively transmitted, but they undergo several rounds of differentiation and proliferation within their intermediate host, the tsetse fly. At each stage, the survival and successful replication of the parasites improve their chances of continuing the life cycle, but little is known about specific molecules that contribute to these processes. Procyclins are the major surface glycoproteins of the insect forms of Trypanosoma brucei. Six genes encode proteins with extensive glutamic acid–proline dipeptide repeats (EP in the single-letter amino acid code), and two genes encode proteins with an internal pentapeptide repeat (GPEET). To study the function of procyclins, we have generated mutants that have no EP genes and only one copy of GPEET. This last gene could not be replaced by EP procyclins, and could only be deleted once a second GPEET copy was introduced into another locus. The EP knockouts are morphologically indistinguishable from the parental strain, but their ability to establish a heavy infection in the insect midgut is severely compromised; this phenotype can be reversed by the reintroduction of a single, highly expressed EP gene. These results suggest that the two types of procyclin have different roles, and that the EP form, while not required in culture, is important for survival in the fly.

Polypyrimidine tract binding protein (PTB) is a major hnRNP protein with multiple roles in mRNA metabolism, including regulation of alternative splicing and internal ribosome entry site-driven translation. We show here that a fourfold overexpression of PTB results in a 75% reduction of mRNA levels produced from transfected gene constructs with different polyadenylation signals (pA signals). This effect is due to the reduced efficiency of mRNA 3 end cleavage, and in vitro analysis reveals that PTB competes with CstF for recognition of the pA signal's pyrimidine-rich downstream sequence element. This may be analogous to its role in alternative splicing, where PTB competes with U2AF for binding to pyrimidine-rich intronic sequences. The pA signal of the C2 complement gene unusually possesses a PTB-dependent upstream sequence, so that knockdown of PTB expression by RNA interference reduces C2 mRNA expression even though PTB overexpression still inhibits polyadenylation. Consequently, we show that PTB can act as a regulator of mRNA expression through both its negative and positive effects on mRNA 3 end processing.Between the branch point and the 3Ј splice site (3ЈSS) of metazoan introns lies a pyrimidine-rich sequence which is critical for efficient splicing (49). Initial analysis of factors that recognize this sequence identified an hnRNP-like protein called polypyrimidine tract binding protein (PTB) or hnRNP I (20,22,23,40). PTB has strong RNA binding activity, since it possesses four tandem RNA recognition motif domains (42). In vitro RNA binding analysis (SELEX) revealed its preferred RNA binding site as UCUU flanked by pyrimidines rather than a nonspecific pyrimidine sequence (41). Subsequent studies indicated that far from being a positively acting splicing factor, PTB actually acts as a selective splicing repressor (55, 57). The splicing factor responsible for recognition of the 3ЈSS pyrimidine tract and AG dinucleotide is the dimeric U2 auxiliary factor protein or U2AF (7). The U2AF 65-kDa subunit interacts with the pyrimidine tract (63), while the smaller 35-kDa subunit directly contacts the 3ЈSS sequence (34). Recognition of the pyrimidine tract by U2AF has been identified as a major site of splicing regulation. This can be modulated in a positive fashion through interaction with splicing-regulatory proteins bound to adjacent exon enhancer sequences (4, 51) or in a negative fashion by competition for binding with PTB (57). In many cases the pyrimidine tract of PTB-regulated exons contains high-affinity binding sites for PTB (41), and direct competition between PTB and U2AF 65 for binding to the pyrimidine tract can lead to exon skipping (28, 50). However, regulation by PTB often requires additional PTB-binding elements remote from the 3ЈSS pyrimidine tract. These may mediate cooperative binding of PTB (11), which can interfere with binding of U2AF as well as other splicing factors (57). Furthermore, PTB exists in several different isoforms generated by alternative splicing (PTB1-referred to throughout the p...

Procyclins are the major surface glycoproteins of insect forms of Trypanosoma brucei. We have previously shown that a conserved 16-mer in the 3 untranslated region (UTR) of procyclin transcripts functions as a positive element in procyclic-form trypanosomes. A systematic analysis of the entire 297-base 3 UTR has now revealed additional elements which are involved in posttranscriptional regulation: a positive element which requires the first 40 bases of the 3 UTR and at least one negative element between nucleotides 101 and 173 (the LII domain). Deletion of either positive element resulted in a >8-fold reduction in the amount of protein but only an ϳ2-fold decrease in the steady-state level of mRNA, suggesting that regulation also occurred at the level of translation. In contrast, deletion of LII caused a threefold increase in the steady-state levels of both the mRNA and protein. LII-16-mer double deletions also gave high levels of expression, suggesting that the 16-mer functions as an antirepressor of the negative element rather than as an independent activator. All three elements have an effect on RNA turnover. When either positive element was deleted, the half-life (t 1/2 ) of the mRNA was reduced from ϳ50 min (the t 1/2 of the wild-type 3 UTR) to <15 min, whereas removal of the LII element resulted in an increased t 1/2 of ϳ100 min. We present a model of posttranscriptional regulation in which the negative domain is counteracted by two positive elements which shield it from nucleases and/or translational repressors.The differentiation of bloodstream forms of Trypanosoma brucei into procyclic forms which replicate in the tsetse fly midgut is marked by the synthesis of a new surface coat composed of procyclins (otherwise known as procyclic acidic repetitive proteins [PARPs]) and the shedding of the variant surface glycoprotein (VSG) coat, which covers the parasites in the mammalian host (31,42,54). It has been estimated that each cell is covered by approximately six million procyclin molecules (9) which are attached to the surface membrane by glycosylphosphatidylinositol (GPI) anchors (15, 16). The parasites divide by binary fission, with a population doubling time of ϳ9 to 10 h in culture, so there is a constant requirement for high levels of procyclin synthesis in order to maintain the density of the coat.Like the majority of genes in trypanosomatids, the procyclin genes form part of polycistronic transcription units (reviewed in reference 50). The trypanosome strain T. brucei 427 contains four procyclin expression sites which are located on separate chromosomes (43). Each expression site consists of tandemly linked procyclin genes (␣ and ␤), followed by a locus-specific procyclin-associated gene (PAG) (6, 28, 51). Two expression sites also contain an additional gene, GRESAG 2 (gene related to ESAG 2), which is very similar to a gene in the VSG expression site (4).When bloodstream form trypanosomes are triggered to differentiate into procyclic forms, there is a 5-to 10-fold increase in transcription initiatio...

Cleavage and polyadenylation (pA) is a fundamental step that is required for the maturation of primary protein encoding transcripts into functional mRNAs that can be exported from the nucleus and translated in the cytoplasm. 3′end processing is dependent on the assembly of a multiprotein processing complex on the pA signals that reside in the pre-mRNAs. Most eukaryotic genes have multiple pA signals, resulting in alternative cleavage and polyadenylation (APA), a widespread phenomenon that is important to establish cell state and cell type specific transcriptomes. Here, we review how pA sites are recognized and comprehensively summarize how APA is regulated and creates mRNA isoform profiles that are characteristic for cell types, tissues, cellular states and disease.

SummaryRNA polymerase II (Pol2) movement through chromatin and the co-transcriptional processing and fate of nascent transcripts is coordinated by transcription elongation factors (TEFs) such as polymerase-associated factor 1 (Paf1), but it is not known whether TEFs have gene-specific functions. Using strand-specific nucleotide resolution techniques, we show that levels of Paf1 on Pol2 vary between genes, are controlled dynamically by environmental factors via promoters, and reflect levels of processing and export factors on the encoded transcript. High levels of Paf1 on Pol2 promote transcript nuclear export, whereas low levels reflect nuclear retention. Strains lacking Paf1 show marked elongation defects, although low levels of Paf1 on Pol2 are sufficient for transcription elongation. Our findings support distinct Paf1 functions: a core general function in transcription elongation, satisfied by the lowest Paf1 levels, and a regulatory function in determining differential transcript fate by varying the level of Paf1 on Pol2.