When oxygen is abundant, quiescent cells efficiently extract energy from glucose primarily by oxidative phosphorylation, whereas under the same conditions tumour cells consume glucose more avidly, converting it to lactate. This long-observed phenomenon is known as aerobic glycolysis 1 , and is important for cell growth 2, 3. Because aerobic glycolysis is only useful to growing cells, it is tightly regulated in a proliferation-linked manner4. Inmammals, this is partly achieved through control of pyruvate kinase isoform expression. The embryonic pyruvate kinase isoform, PKM2, is almost universally re-expressed in cancer2, and promotes aerobic glycolysis, whereas the adult isoform, PKM1, promotes oxidative phosphorylation 2 . These two isoforms result from mutually exclusive alternative splicing of the PKM pre-mRNA, reflecting inclusion of either exon 9 (PKM1) or exon 10 (PKM2). Here we show that three heterogeneous nuclear ribonucleoprotein (hnRNP) proteins, polypyrimidine tract binding protein (PTB, also known as hnRNPI), hnRNPA1 and hnRNPA2, bind repressively to sequences flanking exon 9, resulting in exon 10 inclusion. We also demonstrate that the oncogenic transcription factor c-Myc upregulates transcription of PTB, hnRNPA1 and hnRNPA2, ensuring a high PKM2/PKM1 ratio. Establishing a relevance to cancer, we show that human gliomas overexpress c-Myc, PTB, hnRNPA1 and hnRNPA2 in a manner that correlates with PKM2 expression. Our results thus define a pathway that regulates an alternative splicing event required for tumour cell proliferation.Alternative splicing of PKM has an important role in determining the metabolic phenotype of mammalian cells. The single exon difference imparts the enzymes produced with important functional distinctions. For example, PKM2, but not PKM1, is regulated by the binding of tyrosine phosphorylated peptides, which results in release of the allosteric activator fructose-1-6-bisphosphate and inhibition of pyruvate kinase activity 5 , a property that might allow growth-factor-initiated signalling cascades to channel glycolytic intermediates into biosynthetic processes. The importance of tumour reversion to PKM2 was underscored by experiments in which replacement of PKM2 with PKM1 in tumour cells resulted in markedly reduced growth 2 . Consistent with a critical role in proliferation, re-expression of PKM2 in tumours is robust 2 , although little is known about the regulation of this process. NIH Public Access Author ManuscriptNature. Author manuscript; available in PMC 2010 October 5. We set out to identify RNA binding proteins that might regulate PKM alternative splicing. To this end, we prepared an [α-32 P]UTP-labelled 250-nucleotide RNA spanning the exon 9 (E9) 5′ splice site (EI9), previously identified as inhibitory to E9 inclusion 6 , as well as a labelled RNA from a corresponding region of E10 (EI10) (Fig. 1b), and performed ultraviolet crosslinking assays with HeLa nuclear extracts 7 . After separation by SDS-polyacrylamide gel electrophoresis (PAGE), multiple proteins...

Alternative polyadenylation (APA) is an RNA-processing mechanism that generates distinct 3′ termini on mRNAs and other RNA polymerase II transcripts. It is widespread across all eukaryotic species and is recognized as a major mechanism of gene regulation. APA exhibits tissue specificity and is important for cell proliferation and differentiation. In this Review, we discuss the roles of APA in diverse cellular processes, including mRNA metabolism, protein diversification and protein localization, and more generally in gene regulation. We also discuss the molecular mechanisms underlying APA, such as variation in the concentration of core processing factors and RNA-binding proteins, as well as transcription-based regulation.

Alternative splicing of mRNA precursors provides an important means of genetic control and is a crucial step in the expression of most genes. Alternative splicing markedly affects human development, and its misregulation underlies many human diseases. Although the mechanisms of alternative splicing have been studied extensively, until the past few years we had not begun to realize fully the diversity and complexity of alternative splicing regulation by an intricate protein-RNA network. Great progress has been made by studying individual transcripts and through genome-wide approaches, which together provide a better picture of the mechanistic regulation of alternative premRNA splicing.Alternative splicing is a crucial mechanism for gene regulation and for generating proteomic diversity. Recent estimates indicate that the expression of nearly 95% of human multi-exon genes involves alternative splicing 1,2 . In metazoans, alternative splicing plays an important part in generating different protein products that function in diverse cellular processes, including cell growth, differentiation and death.Splicing is carried out by the spliceosome, a massive structure in which five small nuclear ribonucleoprotein particles (snRNPs) and a large number of auxiliary proteins cooperate to accurately recognize the splice sites and catalyse the two steps of the splicing reaction 1,2 (BOX 1). Spliceosome assembly (BOX 1) begins with the recognition of the 5′ splice site by the snRNP U1 and the binding of splicing factor 1 (SF1) to the branch point 3 and of the U2 auxiliary factor (U2AF) heterodimer to the polypyrimidine tract and 3′ terminal AG 4,5 . This assembly is ATP independent and results in the formation of the E complex, which is converted into the ATP-dependent, pre-spliceosomal A complex after the replacement of SF1 by the U2 snRNP at the branch point. Further recruitment of the U4/U6-U5 tri-snRNP complex leads to the formation of the B complex, which is converted into to the catalytically active C complex after extensive conformational changes and remodelling. Splicing and spliceosome assemblyPre-mRNA splicing is a process in which intervening sequences (introns) are removed from an mRNA precursor. Splicing consists of two transesterification steps, each involving a nucleophilic attack on terminal phosphodiester bonds of the intron. In the first step this is carried out by the 2′ hydroxyl of the branch point (usually adenosine) and in the second step by the 3′ hydroxyl of the upstream (5′) exon 1,2 . This process is carried out in the spliceosome, a dynamic molecular machine the assembly of which involves sequential binding and release of small nuclear ribonucleoprotein particles (snRNPs) and numerous protein factors as well as the formation and disruption of RNA-RNA, protein-RNA and protein-protein interactions.The basic mechanics of spliceosome assembly are well known. Briefly, the process begins with the base pairing of U1 snRNA to the 5′ splice site (ss) and the binding of splicing factor 1 (SF1) to the b...

SR proteins constitute a family of pre-mRNA splicing factors now thought to play several roles in mRNA metabolism in metazoan cells. Here we provide evidence that a prototypical SR protein, ASF/SF2, is unexpectedly required for maintenance of genomic stability. We first show that in vivo depletion of ASF/SF2 results in a hypermutation phenotype likely due to DNA rearrangements, reflected in the rapid appearance of DNA double-strand breaks and high-molecular-weight DNA fragments. Analysis of DNA from ASF/SF2-depleted cells revealed that the nontemplate strand of a transcribed gene was single stranded due to formation of an RNA:DNA hybrid, R loop structure. Stable overexpression of RNase H suppressed the DNA-fragmentation and hypermutation phenotypes. Indicative of a direct role, ASF/SF2 prevented R loop formation in a reconstituted in vitro transcription reaction. Our results support a model by which recruitment of ASF/SF2 to nascent transcripts by RNA polymerase II prevents formation of mutagenic R loop structures.

Summary Alternative polyadenylation (APA) is emerging as a widespread mechanism used to control gene expression. Like alternative splicing, usage of alternative poly(A) sites allows a single gene to encode multiple mRNA transcripts. In some cases, this changes the mRNA coding potential; in other cases, the code remains unchanged but the 3’UTR length is altered, influencing the fate of mRNAs in several ways, for example, by altering the availability of RNA binding protein sites and microRNA binding sites. The mechansims governing both global and gene-specific APA are only starting to be deciphered. Here we review what is known about these mechanisms and the functional consequences of alternative polyadenlyation.