A Carboxy-Terminal Basic Region Controls RNA Polymerase III Transcription Factor Activity of Human La Protein

Abstract

). Others have previously dissected the La protein into an N-terminal domain that binds RNA and a C-terminal domain that does not. Here, deletion and substitution mutants of La were examined for general RNA binding, RNA 3-end protection, and transcription factor activity. Although some La mutants altered in a C-terminal basic region bind RNA in mobility shift assays, they are defective in RNA 3-end protection and do not support transcription, while one C-terminal substitution mutant is defective only in transcription. Moreover, a Cterminal fragment lacking RNA binding activity appears able to support low levels of transcription by pol III. While efficient multiround transcription is supported only by mutants that bind RNA and contain a C-terminal basic region. These analyses indicate that RNA binding contributes to but is not sufficient for La transcription factor activity and that the C-terminal domain plays a role in transcription that is distinguishable from simple RNA binding. The transcription factor activity of La can be reversibly inhibited by RNA, suggesting the potential for feedback inhibition of pol III transcription.Eukaryotic RNA polymerase III (pol III) is responsible for synthesizing the abundant transcripts of tRNA, 5S rRNA, 7SL RNA, and U6 RNA genes as well as other small RNA genes (reviewed in reference 44). To produce a sufficient quantity of RNAs of the correct structure, transcription initiation and termination must be accurate and reinitiation must be efficient. pol III alone cannot accomplish this and must rely on transcription factors (TFs) that bind to control elements and direct RNA synthesis. The adenovirus-associated (VA1) RNA gene and cellular tRNA and Alu genes have internal promoters and 3Ј terminators that direct transcription by pol III. Mammalian TFIIIC1 and TFIIIC2 bind to the control elements of these genes (12, 43). After TFIIIB joins, the resulting preinitiation complex is stable and can be recycled for multiple rounds of transcription by pol III (5,21,27,43).Mammalian in vitro transcription assays have used reconstituted systems that contain at least one partially purified fraction. For example, mammalian TFIIIC2, TFIIIB, and pol III have been highly purified, while TFIIIC1 and TFIIIC1Ј represent relatively crude yet essential fractions (42, 43). In the Saccharomyces cerevisiae system, elegant studies have shown that recombinant or highly purified TFIIIB and TFIIIC are sufficient for multiround transcription (21), although it has also been reported that transcription can be stimulated by TFIIIE, a partially characterized fraction (13, 37). Thus, in both yeast and mammalian pol III systems, while the central factors required for in vitro transcription have been identified and continue to be characterized, evidence also suggests that ancillary, less well characterized factors may stimulate transcription. While remarkable progress has been made in understanding assembly of the preinitiation complex, relatively less effort has been directed toward understanding of the mechanism...

Human La is found at RNA polymerase III-transcribed genes in vivo

“…Immunodepletion of La from cell extracts was found to reduce pol III output in vitro, which led to the suggestion that La could act as a transcriptional termination factor that mediates nascent transcript release (17,18). Indeed, addition of human recombinant La to isolated pol III transcription complexes assembled from mammalian cell extracts led to increases in transcription, apparently due to enhanced pol III recycling and reinitiation (14,19,20). However, a number of other studies contradict these observations.…”

La protein binds the predicted loop structures in the 3′ non-coding region of Japanese encephalitis virus genome: role in virus replication

“…These values are comparable to the affinity with which La protein binds to its known natural ligands in the normal cell. For example, pre-t-RNA Val , hY1 and hY4 RNAs interact with La protein with K d values of 7.3, 6 and 5 nM, respectively (Goodier et al, 1997;Horke et al, 2004). The K d values for La protein interaction with JEV RNA were similar to those obtained for La protein binding to Sindbis virus negativestrand RNA (15.4 nM) (Pardigon & Strauss 1996), human immunodeficiency virus trans-activation response element RNA (17 nM) (Chang et al, 1994), rubella virus RNA (2 nM) (Duncan & Nakhasi 1997) and poliovirus 59 NCR RNA (4 nM) (Meerovitch et al, 1993).…”

Novel CNBP‐ and La‐based translation control systems for mammalian cells

“…La homologues have since been identified in a variety of insect, yeast, and mammalian species (Bai et al, 1994;Chan et al, 1989;Pardigon and Strauss, 1996;Scherly et al, 1993). Although its physiological role has not yet been fully established La has been implied in a variety of functions including (1) initiation and termination of RNA polymerase III-mediated transcription , (2) nuclear RNA export (Simons et al, 1996), (3) interaction with tRNAs, 5S rRNA, U6 small nuclear RNA, 7SL RNAs, cytoplasmic Y RNAs, 4.5S I and 4.5S II RNAs and Alu transcripts (Chambers et al, 1983;Fabini et al, 2000;Hendrick et al, 1981;Lerner et al, 1981;Rinke and Steitz, 1982;1985), (4) chaperoning of small RNAs (Kufel et al, 2000), (5) interaction with 5Ј noncoding regions of viruses (Duncan and Nakhasi, 1997;Goodier et al, 1997;Kurilla and Keene, 1983;Pardigon and Strauss, 1996;Park and Katze, 1995;Svitkin et al, 1994), and (6) translation enhancement of endogenous and viral mRNAs (Ali et al, 2000;Carter and Sarnow, 2000;Crosio et al, 2000;Kim and Jang, 1999;Kim et al, 2001;Waysbort et al, 2001;Zhu et al, 2001).…”