HeLa cytoplasmic extracts contain both 3¢±5¢ and 5¢±3¢ exonuclease activities that may play important roles in mRNA decay. Using an in vitro RNA deadenylation/decay assay, mRNA decay intermediates were trapped using phosphothioate-modi®ed RNAs. These data indicate that 3¢±5¢ exonucleolytic decay is the major pathway of RNA degradation following deadenylation in HeLa cytoplasmic extracts. Immuno-depletion using antibodies speci®c for the exosomal protein PM-Scl75 demonstrated that the human exosome complex is required for ef®cient 3¢±5¢ exonucleolytic decay. Furthermore, 3¢±5¢ exonucleolytic decay was stimulated dramatically by AU-rich instability elements (AREs), implicating a role for the exosome in the regulation of mRNA turnover. Finally, PM-Scl75 protein was found to interact speci®cally with AREs. These data suggest that the interaction between the exosome and AREs plays a key role in regulating the ef®ciency of ARE-containing mRNA turnover.

We have previously demonstrated that PM-Scl-75, a component of the human exosome complex involved in RNA maturation and mRNA decay, can specifically interact with RNAs containing an AU-rich instability element. Through the analysis of a series of deletion mutants, we have now shown that a 266 amino acid fragment representing the RNase PH domain is responsible for the sequence-specific binding to AU-rich elements. Furthermore, we found that the RNase PH domains from two other exosomal components, OIP2 and RRP41, as well as from Escherichia coli polynucleotide phosphorylase, are all capable of specifically interacting with RNAs containing an AU-rich element with similar affinities. Finally, we demonstrate that the interaction of the RNase PH domain of PM-Scl-75 is readily competed by poly(U), but only inefficiently using other homopolymeric RNAs. These data demonstrate that RNase PH domains in general have an affinity for U-and AU-rich sequences, and broaden the potential role in RNA biology of proteins containing these domains.

Eukaryotic transcriptional regulatory signals, defined as core and activator promoter elements, have yet to be identified in the earliest diverging group of eukaryotes, the primitive protozoans, which include the Trypanosomatidae family of parasites. The divergence within this family is highlighted by the apparent absence of the "universal" transcription factor TATA-binding protein. To understand gene expression in these protists, we have investigated spliced leader RNA gene transcription. The RNA product of this gene provides an m 7 G cap and a 39-nucleotide leader sequence to all cellular mRNAs via a trans-splicing reaction. Regulation of spliced leader RNA synthesis is controlled by a tripartite promoter located exclusively upstream from the transcription start site. Proteins PBP-1 and PBP-2 bind to two of the three promoter elements in the trypanosomatid Leptomonas seymouri. They represent the first trypanosome transcription factors with typical doublestranded DNA binding site recognition. These proteins ensure efficient transcription. However, accurate initiation is determined an initiator element with a a loose consensus of CYAC/AYR (؉1), which differs from that found in metazoan initiator elements as well as from that identified in one of the earliest diverging protozoans, Trichomonas vaginalis. Trypanosomes may utilize initiator element-protein interactions, and not TATA sequence-TATA-binding protein interactions, to direct proper transcription initiation by RNA polymerase II.Molecular studies of trypanosomatids, a ubiquitous and diverse family of protozoan pathogens, have revealed strikingly unusual mechanisms of mRNA synthesis. One central device is that two independent transcription events direct each mRNA produced in the trypanosome nucleus (for review, see Ref. 1). The protein-coding portion is transcribed as a single primary mRNA, often containing several open reading frames flanked by 5Ј-and 3Ј-untranslated regions. The capped 5Ј-end portion is transcribed as a short spliced leader (SL) 1 RNA. The two parts are fused in a trans-splicing reaction that yields a functional mRNA. During fusion, the 39 nt present on the 5Ј-end of the SL RNA (and referred to as the SL) are transferred to a region upstream from the coding region on the primary mRNA (2). Addition of the SL provides each mRNA with an m 7 G cap as well as four extensively methylated nucleotides, at positions 1-4 within the 39-nt SL RNA (3).The SL RNA is transcribed from a highly reiterated set of genes. In contrast to the long primary transcripts that form the bulk of the mature mRNA, each SL RNA has a discrete transcriptional start site. ␣-Amanitin studies show that it is very probable, though not proven, that the SL RNA gene is transcribed by RNA polymerase (pol) II. The primary SL RNA transcript and the transcript present in the trans-splicing spliceosome possess identical 5Ј-and 3Ј-ends, indicating that both transcription initiation and termination regulate the accumulation of SL RNA. SL RNA expression has been monitored using independe...

Three types of exonucleases contribute to the turnover of messenger RNAs in eukaryotic cells: (1) general 3'-to-5' exonucleases, (2) poly(A)-specific 3'-to-5' exonucleases, and (3) 5'-to-3' exonucleases. All three of these activities can be detected in cytoplasmic extracts from a variety of eukaryotic cells. In this chapter, we describe the preparation and use of HeLa cytoplasmic S100 extracts to study these three distinct exonuclease activities. Also included is an immunodepletion protocol that can be used to identify the enzyme responsible for a given activity. These protocols can be easily expanded to the study of trans-acting factors, cis-acting RNA sequence elements, and the interplay of components involved in RNA turnover in various mammalian cell types.