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At least 19 recordsLinked to original sources
PubMed2005

U7 snRNP-specific Lsm11 protein: dual binding contacts with the 100 kDa zinc finger processing factor (ZFP100) and a ZFP100-independent function in histone RNA 3' end processing.

The 3' cleavage generating non-polyadenylated animal histone mRNAs depends on the base pairing between U7 snRNA and a conserved histone pre-mRNA downstream element. This interaction is enhanced by a 100 kDa zinc finger protein (ZFP100) that forms a bridge between an RNA hairpin element upstream of the processing site and the U7 small nuclear ribonucleoprotein (snRNP). The N-terminus of Lsm11, a U7-specific Sm-like protein, was shown to be crucial for histone RNA processing and to bind ZFP100. By further analysing these two functions of Lsm11, we find that Lsm11 and ZFP100 can undergo two interactions, i.e. between the Lsm11 N-terminus and the zinc finger repeats of ZFP100, and between the N-terminus of ZFP100 and the Sm domain of Lsm11, respectively. Both interactions are not specific for the two proteins in vitro, but the second interaction is sufficient for a specific recognition of the U7 snRNP by ZFP100 in cell extracts. Furthermore, clustered point mutations in three phylogenetically conserved regions of the Lsm11 N-terminus impair or abolish histone RNA processing. As these mutations have no effect on the two interactions with ZFP100, these protein regions must play other roles in histone RNA processing, e.g. by contacting the pre-mRNA or additional processing factors.

Amino Acid Sequence↗
PubMed2025

Rho-dependent termination and RNase E-mediated cleavage: dual pathways for RNA 3' end processing in polycistronic mRNA.

"Pre-full-length" transcripts are produced at the end of the polycistronic galactose (gal) operon, 5' galE-galT-galK-galM 3', via Rho-dependent transcription termination (RDT) and -independent transcription termination. The 3' end of the full-length galETKM mRNA is acquired by exonucleolytic processing of the 3'-OH ends of the pre-full-length transcripts. However, the gal operon produces an mRNA termed galE whose 3' end forms approximately 120 nucleotides downstream of the galE stop codon, within the subsequent gene, galT, thereby establishing polarity in gene expression. In this study, we investigated the molecular processes that generate the 3' end of galE mRNA. We discovered that the 3' ends of pre-galE mRNA are produced in the middle of galT as a result of the combination of two separate molecular processes-one previously reported as RDT and the other as unreported RNase E-mediated transcript cleavage. The 3' ends of pre-galE mRNA undergo exonucleolytic processing to the 3' end of galE mRNA observed in vivo. A hairpin structure containing an 8 bp stem and a 4-nucleotide loop, located 5-10 nucleotides upstream of the 3' ends of galE mRNA, blocks exoribonuclease digestion and renders transcript stability. These findings demonstrate that RNase E-contrary to its general role in mRNA degradation-produces RNA 3' ends that regulate polarity in gene expression.IMPORTANCEThis study reports the findings of two molecular mechanisms that generate the 3' ends of pre-galE mRNA in the gal operon, viz., Rho-dependent transcription termination and RNase E-mediated cleavage. These 3' ends are subsequently processed to produce stable galE mRNA with a hairpin structure that prevents exoribonuclease degradation. This mechanism establishes gene expression polarity by generating the 3' end of galE mRNA within galT in contrast to the usual mRNA degradation role of RNase E. The study reveals a unique role of RNase E in mRNA processing and stability.

RNA, Messenger↗
PubMed1997

Chloroplast endoribonuclease p54 involved in RNA 3'-end processing is regulated by phosphorylation and redox state.

Chloroplast RNA-binding protein p54 is an endoribonuclease required for 3'end-processing of plastid precursor transcripts. We find that purified p54 can serve as a phosphate acceptor for protein kinases in vitro. Both the processing and RNA-binding activities of p54 are enhanced by phosphorylation and decreased by dephosphorylation. In addition, the enzyme is activated by the oxidized form of glutathione and inhibited by the reduced form, whereas other redox reagents that were tested showed no effect. Kinase treatment of p54 prior to oxidation by glutathione resulted in highest levels of activation, suggesting that phosphorylation and redox state act together to control p54 activity in vitro and possibly also in vivo.

Chloroplast Proteins↗
PubMed1991

An RNA secondary structure juxtaposes two remote genetic signals for human T-cell leukemia virus type I RNA 3'-end processing.

Sequence analysis of the human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR) does not reveal a polyadenylation consensus sequence, AAUAAA, close to the polyadenylation site at the 3' end of the viral RNA. Using site-directed mutagenesis, we demonstrated that two cis-acting signals are required for efficient RNA processing in HTLV-I LTR: (i) a remote AAUAAA hexamer at a distance of 276 nucleotides upstream of the polyadenylation site, and (ii) the 20-nucleotide GU-rich sequence immediately downstream from the poly(A) site. It has been postulated that the folding of RNA into a secondary structure juxtaposes the AAUAAA sequence, in a noncontiguous manner, to within 14 nucleotides of the polyadenylation site. To test this hypothesis, we introduced deletions and point mutations within the U3 and R regions of the LTR. RNA 3'-end processing occurred efficiently at the authentic HTLV-I poly(A) site after deletion of the sequences predicted to form the secondary structure. Thus, the genetic analysis supports the hypothesis that folding of the HTLV-I RNA in the U3 and R regions juxtaposes the AAUAAA sequence and the poly(A) site to the correct functional distance. This unique arrangement of RNA-processing signals is also found in the related retroviruses HTLV-II and bovine leukemia virus.

Base Sequence↗
PubMed2004

A novel function for Sam68: enhancement of HIV-1 RNA 3' end processing.

Both cis elements and host cell proteins can significantly affect HIV-1 RNA processing and viral gene expression. Previously, we determined that the exon splicing silencer (ESS3) within the terminal exon of HIV-1 not only reduces use of the adjacent 3' splice site but also prevents Rev-induced export of the unspliced viral RNA to the cytoplasm. In this report, we demonstrate that loss of unspliced viral RNA export is correlated with the inhibition of 3' end processing by the ESS3. Furthermore, we find that the host factor Sam68, a stimulator of HIV-1 protein expression, is able to reverse the block to viral RNA export mediated by the ESS3. The reversal is associated with a stimulation of 3' end processing of the unspliced viral RNA. Our findings identify a novel activity for the ESS3 and Sam68 in regulating HIV-1 RNA polyadenylation. Furthermore, the observations provide an explanation for how Sam68, an exclusively nuclear protein, modulates cytoplasmic utilization of the affected RNAs. Our finding that Sam68 is also able to enhance 3' end processing of a heterologous RNA raises the possibility that it may play a similar role in regulating host gene expression.

3' Untranslated Regions↗
PubMed2003

Contribution of domain structure to the RNA 3' end processing and degradation functions of the nuclear exosome subunit Rrp6p.

The 3'-5' riboexonuclease Rrp6p, a nuclear component of the exosome, functions with other exosome components to produce the mature 3' ends of 5.8S rRNA, sno- and snRNAs, and to destroy improperly processed precursor (pre)-rRNAs and pre-mRNAs. Rrp6p is a member of the RNase D family of riboexonucleases and displays a high degree of homology with the active site of the deoxyriboexonuclease domain of Escherichia coli DNA polymerase I, the crystal structure of which indicates a two-metal ion mechanism for phosphodiester bond hydrolysis. Mutation of each of the conserved residues predicted to coordinate metal ions in the active site of Rrp6p abolished activity of the enzyme in vitro and in vivo. Complete loss of Rrp6p activity caused by the Y361F and Y361A mutations supports the critical role proposed for the phenolic hydroxyl of Tyr361 in the reaction mechanism. Rrp6p also contains an helicase RNase D C-terminal (HRDC) domain of unknown function that is similar to domains in the Werner's and Bloom's Syndrome proteins. A point mutation in this domain results in Rrp6p that localizes to the nucleus, but fails to efficiently process the 3' ends of 5.8S pre-rRNA and some pre-snoRNAs. In contrast, this mutant retains the ability to degrade rRNA processing intermediates and 3'-extended, poly(A)+ snoRNAs. These findings indicate the potential for independent control of the processing and degradation functions of Rrp6p.

Amino Acid Sequence↗
PubMed2003

FY is an RNA 3' end-processing factor that interacts with FCA to control the Arabidopsis floral transition.

The nuclear RNA binding protein, FCA, promotes Arabidopsis reproductive development. FCA contains a WW protein interaction domain that is essential for FCA function. We have identified FY as a protein partner for this domain. FY belongs to a highly conserved group of eukaryotic proteins represented in Saccharomyces cerevisiae by the RNA 3' end-processing factor, Pfs2p. FY regulates RNA 3' end processing in Arabidopsis as evidenced through its role in FCA regulation. FCA expression is autoregulated through the use of different polyadenylation sites within the FCA pre-mRNA, and the FCA/FY interaction is required for efficient selection of the promoter-proximal polyadenylation site. The FCA/FY interaction is also required for the downregulation of the floral repressor FLC. We propose that FCA controls 3' end formation of specific transcripts and that in higher eukaryotes, proteins homologous to FY may have evolved as sites of association for regulators of RNA 3' end processing.

Amino Acid Sequence↗
PubMed2002

Functional interaction of yeast pre-mRNA 3' end processing factors with RNA polymerase II.

The RNA polymerase II CTD is essential for 3' end cleavage of metazoan pre-mRNAs and binds 3' end processing factors in vitro. We show genetic and biochemical interactions between the CTD and the Pcf11 subunit of the yeast cleavage/polyadenylation factor, CFIA. In vitro binding to Pcf11 required phosphorylation of the CTD on Ser2 in the YSPTSPS heptad repeats. Deletion of the yeast CTD reduced the efficiency of cleavage at poly(A) sites, and the length of poly(A) tails suggesting that it helps couple 3' end formation with transcription. Consistent with this model, the 3' end processing factors CFIA, CFIB, and PFI were recruited to genes progressively, starting at the 5' end, in a process that required ongoing transcription.

Animals↗
PubMed1996

Length suppression in histone messenger RNA 3'-end maturation: processing defects of insertion mutant premessenger RNAs can be compensated by insertions into the U7 small nuclear RNA.

Efficient 3'-end processing of cell cycle-regulated mammalian histone premessenger RNAs (pre-mRNAs) requires an upstream stem-loop and a histone downstream element (HDE) that base pairs with the U7 small ribonucleoprotein. Insertions between these elements have two effects: the site of cleavage moves in concert with the HDE and processing efficiency declines. We used Xenopus oocytes to ask whether compensatory length insertions in the human U7 RNA could restore the fidelity and efficiency of processing of mouse histone insertion pre-mRNAs. An insertion of 5 nt into U7 RNA that extends its complementary to the HDE compensated for both defects in processing of a 5-nt insertion substrate; a noncomplementary insertion into U7 did not. Yet, the noncomplementary insertion mutant U7 was shown to be active on insertion substrates further mutated to allow base pairing. Our results suggest that the histone pre-mRNA becomes rigidified upstream of its HDE, allowing the bound U7 small ribonucleoprotein to measure from the HDE to the cleavage site. Such a mechanism may be common to other RNA measuring systems. To our knowledge, this is the first demonstration of length suppression in an RNA processing system.

Animals↗
PubMed2002

RNA 3' readthrough of oncoretrovirus and lentivirus: implications for vector safety and efficacy.

The expression of reporter genes driven by the same human elongation factor 1alpha (EF1alpha) promoter in murine leukemia virus (MLV)- and human immunodeficiency virus type 1 (HIV-1)-based vectors was studied in either transfected or virally transduced cells. The HIV-1 vectors consistently expressed 3 to 10 times higher activity than the MLV vectors at both the RNA and protein levels. The difference was not attributable to transcriptional interference, alternative enhancer/silencer, or differential EF1alpha intron splicing. Based on nuclear run-on assays, both vectors exhibited similar EF1alpha transcriptional activity. The reduced RNA levels of MLV vectors could not be explained by the decrease in RNA half-lives. Southern analysis of proviral DNA indicated that both HIV-1 and MLV vectors efficiently propagated the EF1alpha intron in the transduced cells. To decipher the discrepancy in transgene expression between MLV and HIV-1 vectors, the role of RNA 3'-end processing was examined using a sensitive Cre/lox reporter assay. The results showed that MLV vectors, but not HIV-1 vectors, displayed high frequencies of readthrough of the 3' polyadenylation signal. Interestingly, the polyadenylation signal of a self-inactivating (SIN) HIV-1 vector was as leaky as that of the MLV vectors, suggesting a potential risk of oncogene activation by the lentiviral SIN vectors. Together, our results suggest that an efficient polyadenylation signal would improve both the efficacy and the safety of these vectors.

Animals↗
PubMed2004

Antisense transcript and RNA processing alterations suppress instability of polyadenylated mRNA in chlamydomonas chloroplasts.

In chloroplasts, the control of mRNA stability is of critical importance for proper regulation of gene expression. The Chlamydomonas reinhardtii strain Delta26pAtE is engineered such that the atpB mRNA terminates with an mRNA destabilizing polyadenylate tract, resulting in this strain being unable to conduct photosynthesis. A collection of photosynthetic revertants was obtained from Delta26pAtE, and gel blot hybridizations revealed RNA processing alterations in the majority of these suppressor of polyadenylation (spa) strains, resulting in a failure to expose the atpB mRNA 3' poly(A) tail. Two exceptions were spa19 and spa23, which maintained unusual heteroplasmic chloroplast genomes. One genome type, termed PS+, conferred photosynthetic competence by contributing to the stability of atpB mRNA; the other, termed PS-, was required for viability but could not produce stable atpB transcripts. Based on strand-specific RT-PCR, S1 nuclease protection, and RNA gel blots, evidence was obtained that the PS+ genome stabilizes atpB mRNA by generating an atpB antisense transcript, which attenuates the degradation of the polyadenylated form. The accumulation of double-stranded RNA was confirmed by insensitivity of atpB mRNA from PS+ genome-containing cells to S1 nuclease digestion. To obtain additional evidence for antisense RNA function in chloroplasts, we used strain Delta26, in which atpB mRNA is unstable because of the lack of a 3' stem-loop structure. In this context, when a 121-nucleotide segment of atpB antisense RNA was expressed from an ectopic site, an elevated accumulation of atpB mRNA resulted. Finally, when spa19 was placed in a genetic background in which expression of the chloroplast exoribonuclease polynucleotide phosphorylase was diminished, the PS+ genome and the antisense transcript were no longer required for photosynthesis. Taken together, our results suggest that antisense RNA in chloroplasts can protect otherwise unstable transcripts from 3'-->5' exonuclease activity, a phenomenon that may occur naturally in the symmetrically transcribed and densely packed chloroplast genome.

Animals↗
PubMed1991

Leader region of mdg1 Drosophila retrotransposon RNA contains 3'-end processing sites.

Transient expression of the mdg1 deletion mutants revealed sites of 3'-end processing in the leader region of the transcribed RNA. The efficiency of the processing is regulated in different types of cells. The sequences within the mdg1 body and the 3'-LTR are involved in its regulation. We have also shown, that one of the small open reading frames in the mdg1 leader region in principle might be translated.

Animals↗
PubMed2002

A 3'-terminal minihelix in the precursor of human spliceosomal U2 small nuclear RNA.

U2 RNA is one of five small nuclear RNAs that participate in the majority of mRNA splicing. In addition to its role in mRNA splicing, the biosynthesis of U2 RNA and three of the other spliceosomal RNAs is itself an intriguing process involving nuclear export followed by 5'-cap hypermethylation, assembly with specific proteins, 3' end processing, and then nuclear import. Previous work has identified sequences near the 3' end of pre-U2 RNA that are required for accurate and efficient processing. In this study, we have investigated the structural basis of U2 RNA 3' end processing by chemical and enzymatic probing methods. Our results demonstrate that the 3' end of pre-U2 RNA is a minihelix with an estimated stabilization free energy of -6.9 kcal/mol. Parallel RNA structure mapping experiments with mutant pre-U2 RNAs revealed that the presence of this 3' minihelix is itself not required for in vitro 3'-processing of pre-U2 RNA, in support of earlier studies implicating internal regions of pre-U2 RNA. Other considerations raise the possibility that this distinctive structural motif at the 3' end of pre-U2 RNA plays a role in the cleavage of the precursor from its longer primary transcript or in its nucleocytoplasmic traffic.

Base Sequence↗
PubMed2005

A Requirement for the Saccharomyces cerevisiae Paf1 complex in snoRNA 3' end formation.

RNA synthesis and processing are coordinated by proteins that associate with RNA polymerase II (pol II) during transcription elongation. The yeast Paf1 complex interacts with RNA pol II and mediates histone modifications during elongation. To elucidate the functions of this complex, we isolated missense mutations in the gene encoding the Rtf1 subunit and used them to identify functionally interacting proteins. We identified NAB3 as a dosage suppressor of rtf1. Nab3, together with Nrd1, directs 3' end formation of nonpolyadenylated RNA pol II transcripts, such as snoRNAs. Deletion of Paf1, but not the Set1, Set2, or Dot1 histone methyltransferases, causes accumulation of snoRNA transcripts that are extended at their 3' ends. The Paf1 complex associates with and facilitates Nrd1 recruitment to the SNR47 gene, suggesting a direct involvement in 3' end formation. Our results reveal a posttranscriptional function for the Paf1 complex, which appears unrelated to its role in histone methylation.

Gene Expression Regulation↗
PubMed1987

Identification of a complex associated with processing and polyadenylation in vitro of herpes simplex virus type 1 thymidine kinase precursor RNA.

Cleavage and polyadenylation of substrate RNAs containing the herpes simplex virus type 1 (HSV-1) thymidine kinase (tk) gene polyadenylation signal region were examined in HeLa cell nuclear extract. 3'-End RNA processing was accurate and efficient and required ATP and Mg2+. Cleavage, but not polyadenylation, occurred in the presence of EDTA or when ATP was replaced with 3' dATP (cordycepin) or AMP(CH2)PP, a nonhydrolyzable analog of ATP. Processing in vitro and in vivo showed the same signal element requirements: a series of substrates containing linker scanning, internal deletion, and small insertion mutations was processed with the same relative efficiencies and at the same sites in vitro and in vivo. A complex involved in 3'-end RNA processing was identified by gel mobility shift analysis. This complex formed rapidly, reached a maximum level after 20 to 30 min, and was much reduced after 2 h. Very little complex was formed at 0 degree C or with substrates lacking a polyadenylation signal. Entry of 32P-labeled tk substrate into the complex could be prevented by addition of excess 35S-labeled tk or adenovirus L3 precursor RNAs. Competition was not observed with tk RNAs lacking a complete polyadenylation signal.

Adenosine Triphosphate↗
PubMed2003

The 3' ends of human pre-snRNAs are produced by RNA polymerase II CTD-dependent RNA processing.

Proper 3' end formation of the human pre-snRNAs synthesized by pol II requires the cis-acting 3' box, although the precise function of this element has proved difficult to determine. In vivo, 3' end formation is tightly linked to transcription. However, we have now been able to obtain transcription-independent 3' box-dependent processing in vitro. This finally demonstrates that the 3' end of pre-snRNAs is produced by RNA processing rather than by termination of transcription. The phosphorylated form of the C-terminal domain (CTD) of pol II activates the processing event in vitro, consistent with our previous demonstration of the role of the CTD in pre-snRNA 3' end formation in vivo. In addition, we show that sequences upstream from the 3' box of the U2 snRNA gene influence 3' end formation both in vivo and in vitro.

Base Sequence↗
PubMed2002

The 3' end formation in small RNAs.

Small RNAs are a major class of RNAs along with transfer RNAs, ribosomal RNAs, and messenger RNAs. They vary in size from less than 100 nucleotides to several thousand nucleotides and have been identified and characterized both in prokaryotes and eukaryotes. Small RNAs participate in a variety of cellular functions including regulating RNA synthesis, RNA processing, guiding modifications in RNA, and in transport of proteins. Small RNAs are generated by a series of posttranscriptional processing steps following transcription. While RNA 5' end structure, 5' cap formation, and RNA processing mechanisms have been fairly well characterized, the 3' end processing is poorly understood. Recent data point to an emerging theme in small RNAs metabolism in which the 3' end processing is mediated by the exosome, a large multienzyme complex. In addition to removal of nucleotides by the exosome, there is simultaneous rebuilding of the 3' end of some small RNA by adenylation and/or uridylation. This review presents a picture of both degradative and rebuilding reactions operative on the 3' end of some small RNA molecules in prokaryotes and eukaryotes.

Animals↗
PubMed1987

Transcription termination and RNA processing in the 3'-end spacer of mouse ribosomal RNA genes.

The 3' termini of ribosomal RNA precursors from mouse FM3A cultured cells are mapped to eight sites within 625 bp downstream from the 3' terminus of 28 S rRNA. Three additional sites are mapped in liver RNA from C3H/He strain mice. Two of them, the sites at 570 bp and 625 bp are assumed to be termination sites in vivo, because they correspond to in vitro termination sites of RNA polymerase I, and 45 S RNAs having these 3' termini decay with kinetics distinct from others. The amount of 45 S RNA having the 3' terminus at other sites is variable among several mouse strains, despite their having the same DNA sequence in these regions. The ability to produce 3' termini in these sites seems to follow Mendel's law of inheritance. Therefore, we postulate that these nine sites are RNA processing sites which are controlled genetically.

Animals↗