A meeting “Ribosome Biogenesis and Nucleolar Function” was held at Lake Tahoe, California, August 17–21, 2000, ably organized by Lasse Lindahl and Janice Zengel (University of Maryland, Baltimore County), together with Maurille Fournier (University of Massachusetts), Craig Pikaard (Washington University), and John Woolford (Carnegie-Mellon University). The meeting was noteworthy in several respects. First, it occurred within only a few days (on either side) of the publication of breathtaking 2.4–3.3 Å structures of the large and small ribosomal subunits. Second, it addressed how this machine is built in the cell to a greater extent than most previous ribosome meetings. Third, the meeting congratulated Masayasu Nomura for his important, and still continuing, contributions to the ribosome field.

Translational fidelity, programmed frameshifting, and nonsense-mediated decay (NMD) may require common proteins, reflecting related events in translation and ribosome function (reviewed in Czaplinski et al., 1999). This letter focuses on the possible link between programmed frameshifting and NMD, and seeks to reconcile apparently conflicting conclusions concerning a proposed overlap in the factors involved.

DNA harvested directly from complex natural microbial communities by PCR has been successfully used to predict RNase P RNA structure, and can potentially provide an abundant source of information for structural predictions of other RNAs. In this study, we utilized genetic variation in natural communities to test and refine the secondary and tertiary structural model for the bacterial tmRNA. The variability of proposed tmRNA secondary structures in different organisms and the lack of any predicted tertiary structure suggested that further refinement of the tmRNA could be useful. To increase the phylogenetic representation of tmRNA sequences, and thereby provide additional data for statistical comparative analysis, we amplified, sequenced, and compared tmRNA sequences from natural microbial communities. Using primers designed from gamma proteobacterial sequences, we determined 44 new tmRNA sequences from a variety of environmental DNA samples. Covariation analyses of these sequences, along with sequences from cultured organisms, confirmed most of the proposed tmRNA model but also provided evidence for a new tertiary interaction. This approach of gathering sequence information from natural microbial communities seems generally applicable in RNA structural analysis.

The majority of mRNA turnover is mediated either by mRNA decapping/5′-to-3′ decay or exosome-mediated 3′-to-5′ exonucleolytic decay. Current assays to assess mRNA decapping in vitro using cap-labeled RNA substrates rely on one-dimensional thin layer chromatography. This approach does not, however, resolve free phosphate from 7meGDP, the product of Dcp1p-mediated mRNA decapping. This can result in misinterpretation of the levels of mRNA decapping due to the generation of free phosphate following the action of the unrelated scavenger decapping activity on the products of exosome-mediated decay. In this report, we describe a simple denaturing acrylamide gel-based assay that faithfully resolves all of the possible products that can be generated from cap-labeled RNA substrates by turnover enzymes present in cell extracts. This approach allows a one-step assay to quantitatively assess the contributions of the exosome and DCP-1-type decapping on turnover of an RNA substrate in vitro. We have applied this assay to recalculate the effect of competition of cap-binding proteins on decapping in yeast. In addition, we have used the assay to confirm observations made on regulated mRNA decapping in mammalian extracts that contain much higher levels of exosome activity than yeast extracts.

The poly(A)-limiting element (PLE) is a cis-acting sequence that acts to limit poly(A) tail length on pre-mRNA to <20 nt. Functional PLEs are present in a number of genes, underscoring the generality of this control mechanism. The current study sought to define further the position requirements for poly(A) length regulation and the core sequence that comprises a PLE. Increasing the spacing between the PLE and the upstream 3′ splice site or between the PLE and the downstream AAUAAA had no effect on poly(A) length control. However, moving the PLE from the terminal exon to either an upstream exon or intron eliminated poly(A) length control. Poly(A) length control was further evaluated using a battery of constructs in which the PLE was maintained in the terminal exon, but where upstream introns were either deleted, modified, or replaced with a polypyrimidine tract. Poly(A) length control was retained in all cases, indicating that the key feature is the presence of the PLE in the terminal exon. A battery of mutations demonstrated the importance of the 5′ pyrimidine-rich portion of the element. Finally, UV crosslinking experiments identified an ∼62-kDa protein in Hela nuclear extract that binds to a wild-type 23-nt PLE RNA oligonucleotides but not to a mutated nonfunctional form of the element.

Posttranscriptional silencing of basic β-1,3-glucanase genes in the tobacco line T17 is manifested by reduced transcript levels of the gn1 transgene and homologous, endogenous basic β-1,3-glucanase genes. An RNA ligation-mediated rapid amplification of cDNA ends (RLM-RACE) technique was used to compare the 3′ termini of gn1 RNAs present in expressing (hemizygous and young homozygous) and silenced (mature homozygous) T17 plants. Full-length, polyadenylated gn1 transcripts primarily accumulated in expressing plants, whereas in silenced T17 plants, mainly 3′-truncated, nonpolyadenylated gn1 RNAs were detected. The relative abundance of these 3′-truncated gn1 RNA species gradually increased during the establishment of silencing in homozygous T17 plants. Similar 3′-truncated, nonpolyadenylated gn1 RNA products were observed in an independent case of β-1,3-glucanase posttranscriptional gene silencing. This suggests that these 3′-truncated gn1 RNAs are a general feature of tobacco plants showing posttranscriptional silencing of the gn1 transgene.

There are several lines of evidence that both RNA viruses and retroviruses recombine according to a copy choice mechanism. Using the brome mosaic virus (BMV)-based system, we recognized elements in the RNA structure that enhance nonhomologous crossovers within or near the local heteroduplex formed by recombining molecules. The same structural motifs were employed in vitro to test the ability of human immunodeficiency virus reverse transcriptase (HIV-RT) to switch templates during DNA synthesis. We demonstrated that a specific combination of the local double-stranded region with short homologous sequences and a hairpin structure allows template switching by HIV-RT. In contrast to BMV replicase, HIV-RT does not mediate the detectable level of recombination using only the heteroduplex structure, though local hybridization between RNA molecules efficiently pauses primer extension. Moreover, the presented data suggest that a proper arrangement of identified structural motifs can ensure site specificity of RNA–RNA recombination. These results indicate that HIV-RT utilizes the same or a very similar mechanism as BMV replicase to change nonhomologous RNA templates in a site-specific manner.

This is in reference to the paper by Nitta et al. published in RNA in March, 1998.

We developed a two-step purification of mammalian polyadenylation complexes assembled in vitro. Biotinylated pre-mRNAs containing viral or immunoglobulin poly(A) sites were incubated with nuclear extracts prepared from mouse myeloma cells under conditions permissive for in vitro cleavage and polyadenylation and the mixture was fractionated by gel filtration; complexes containing biotinylated pre-mRNA and bound proteins were affinity purified on avidin-agarose resin. Western analysis of known components of the polyadenylation complex demonstrated copurification of polyadenylation factors with poly(A) site-containing RNA but not with control RNA substrates containing either no polyadenylation signals or a point mutation of the AAUAAA polyadenylation signal. Polyadenylation complexes that were assembled on exogenous RNA eluted from the Sephacryl column in fractions consistent with their size range extending from 2 to 4 × 106Mr. Complexes endogenous to the extract were of approximately the same apparent size, but more heterogeneous in distribution. This method can be used to study polyadenylation/cleavage complexes that may form upon a number of different RNA sequences, an important step towards defining which factors might differentially associate with specific RNAs.

The 3′ major domain of Escherichia coli 16S rRNA, which occupies the head of the small ribosomal subunit, is involved in several functions of the ribosome. We have used a site-specific crosslinking procedure to gain further insights into the higher-order structure of this domain. Circularly permuted RNAs were used to introduce an azidophenacyl group at specific positions within the 3′ major domain. Crosslinks were generated in a high-ionic strength buffer that has been used for ribosome reconstitution studies and so enables the RNA to adopt a structure recognized by ribosomal proteins. The crosslinking sites were identified by primer extension and confirmed by assessing the mobility of the crosslinked RNA lariats in denaturing polyacrylamide gels. Eight crosslinks were characterized. Among them, one crosslink demonstrates that helix 28 is proximal to the top of helix 34, and two others show that the 1337 region, located in an internal loop at the junction of helices 29, 30, 41, and 42, is proximal to the center of helix 30 and to a segment connecting helix 28 to helix 29. These relationships of vicinity have previously been observed in native 30S subunits, which suggests that the free domain adopts a conformation similar to that within the 30S subunit. Furthermore, crosslinks were obtained in helix 34, which suggest that the upper and lower portions of this helix are in close proximity.

The aim of the present study was to identify functional antisense oligodeoxynucleotides (ODNs) against the rat glutathione S-transferase Mu (GSTM) isoforms, GSTM1 and GSTM2. These antisense ODNs would enable the study of the physiological consequences of GSTM deficiency. Because it has been suggested that the effectiveness of antisense ODNs is dependent on the secondary mRNA structures of their target sites, we made mRNA secondary structure predictions with two software packages, Mfold and STAR. The two programs produced only marginally similar structures, which can probably be attributed to differences in the algorithms used. The effectiveness of a set of 18 antisense ODNs was evaluated with a cell-free transcription/translation assay, and their activity was correlated with the predicted secondary RNA structures. Four phosphodiester ODNs specific for GSTM1, two ODNs specific for GSTM2, and four ODNs targeted at both GSTM isoforms were found to be potent, sequence-specific, and RNase H-dependent inhibitors of protein expression. The IC50 value of the most potent ODN was approximately 100 nM. Antisense ODNs targeted against regions that were predicted by STAR to be predominantly single stranded were more potent than antisense ODNs against double-stranded regions. Such a correlation was not found for the Mfold prediction. Our data suggest that simulation of the local folding of RNA facilitates the discovery of potent antisense sequences. In conclusion, we selected several promising antisense sequences, which, when synthesized as biologically stable oligonucleotides, can be applied for study of the physiological impact of reduced GSTM expression.

Ribosome-inactivating proteins (RIPs) are RNA-N-glycosidases widely present in plants that depurinate RNA in ribosomes at a specific universally conserved position, A4324, in the rat 28S rRNA. A small group of RIPs (cofactor-dependent RIPs) require ATP and tRNA to reach maximal activity on isolated ribosomes. Among cofactor-dependent RIPs, gelonin is specifically and uniquely stimulated by tRNATrp. The active species are avian (chicken) and mammalian (beef, rat, and rabbit) tRNATrp, whereas yeast tRNATrp is completely devoid of stimulating activity. In the present article, bovine and yeast tRNATrp with unmodified bases were prepared by assembly of the corresponding genes from synthetic oligonucleotides followed by PCR and T7 RNA polymerase transcription of the amplified products. The two synthetic tRNAs were fully active (bovine) or inactive (yeast) as the wild-type tRNAs. Construction of chimeric tRNATrp transcripts identified the following bovine nucleotides as recognition elements for gelonin-stimulating activity: G26 and bp G12-C23 in the D arm and G57, A59, and bp G51-C63 and U52-A62 in the T arm. Among single-stranded nucleotides, A59 has a prominent role, but full expression of the gelonin-stimulating activity requires an extensive cooperation between nucleotides in both arms.

Current in vitro assays for RNA editing in kinetoplastids directly examine the products generated by incubation of pre-mRNA substrate with guide RNA (gRNA) and mitochondrial (mt) extract. RNA editing substrates that are modeled on hammerhead ribozymes were designed with catalytic cores that contained or lacked additional uridylates (Us). They proved to be sensitive reporters of editing activity when used for in vitro assays. A deletion editing substrate that is based on A6 pre-mRNA had no ribozyme activity, but its incubation with gRNA and mt extract resulted in its deletion editing and production of a catalytically active ribozyme. Hammerhead ribozymes are thus sensitive tools to assay in vitro RNA editing.

During splicing of nuclear pre-mRNAs, the first step liberates the 5′ exon (exon 1) and yields a lariat intron-3′exon (intron-exon 2) intermediate. The second step results in exon ligation. Previous results indicated that severe truncations of the 5′ exon of the actin pre-mRNA result in a block to the second splicing step in vitro in yeast extracts, leading to an accumulation of intron-exon 2 lariat intermediates. We show that exogenous exon 1 RNA oligonucleotides can chase these stalled intermediates into lariat intron and spliced exons. This reaction requires some of the cis elements and trans-acting factors that are required for a normal second step. There is no strong sequence requirement for the exon 1 added in trans, but oligonucleotides with complementarity to the U5 snRNA conserved loop perform the chase more efficiently. Using a dominant negative mutant of the DEAH-box ATPase Prp16p and ATP depletion, we show that the stalled intermediate is blocked after the Prp16p-dependent step. These results show that exogenous RNAs with various sequences but containing no splicing signals can be incorporated into spliceosomes and undergo RNA recombination and exon shuffling during the second step of pre-mRNA splicing.

Domain V of Escherichia coli 23 S rRNA (residues 2023–2630) was replaced by that from Staphylococcus aureus, thereby introducing 132 changes in the rRNA sequence. The resulting ribosomal mutant was unable to support cell growth. The mutant was rescued, however, by restoring an interaction between domains IV and V (residues 1782 and 2586). Although the importance of this interaction, U/U in E. coli, C/C in S. aureus, is therefore demonstrated, it cannot be the only tertiary interaction important for ribosomal function as the rescued hybrid grew more slowly than the wild type. Additionally, although the single-site mutations U1782C and U2586C in E. coli are viable, the double mutant is lethal.

The rat β-tropomyosin (β-TM) gene encodes both skeletal muscle β-TM mRNA and nonmuscle TM-1 mRNA via alternative RNA splicing. This gene contains eleven exons: exons 1–5, 8, and 9 are common to both mRNAs; exons 6 and 11 are used in fibroblasts as well as in smooth muscle, whereas exons 7 and 10 are used in skeletal muscle. Previously we demonstrated that utilization of the 3′ splice site of exon 7 is blocked in nonmuscle cells. In this study, we use both in vitro and in vivo methods to investigate the regulation of the 5′ splice site of exon 7 in nonmuscle cells. The 5′ splice site of exon 7 is used efficiently in the absence of flanking sequences, but its utilization is suppressed almost completely when the upstream exon 6 and intron 6 are present. The suppression of the 5′ splice site of exon 7 does not result from the sequences at the 3′ end of intron 6 that block the use of the 3′ splice site of exon 7. However, mutating two conserved nucleotides GU at the 5′ splice site of exon 6 results in the efficient use of the 5′ splice site of exon 7. In addition, a mutation that changes the 5′ splice site of exon 7 to the consensus U1 snRNA binding site strongly stimulates the splicing of exon 7 to the downstream common exon 8. Collectively, these studies demonstrate that 5′ splice site competition is responsible, in part, for the suppression of exon 7 usage in nonmuscle cells.

The structure of human 40S ribosomal subunits has been probed by a cross-linking strategy based on the use of oligonucleotide derivatives that modify proteins in the vicinity of specific 18S rRNA sequences. The oligonucleotide derivatives carried a p-azidoperfluorobenzamide group at the 5′ ends and were complementary to 18S rRNA sequences 609–618 and 1047–1061, homologous to the highly conserved regions designated as the “530 stem-loop” and “790 stem-loop”, respectively, in Escherichia coli 16S rRNA. Ribosomal proteins surrounding these sequences were the main targets of the cross-linking. Proteins S3 and S5 were cross-linked to the derivative complementary to the sequence 609–618, and proteins S2 and S3 were modified by the derivative complementary to the sequence 1047–1061. Cross-linking was not affected by binding of 40S subunits to either poly(U) or poly(U) and Phe-tRNAPhe.

Altman and colleagues (this issue) call attention to the inability of current standardized enzyme nomenclature to distinguish between enzymatic activities that reside in nonhomologous macromolecules. This issue is highlighted by the fact that the pre-tRNA 5′-maturation activities of bacteria and plant chloroplasts present the first instance (of which I am aware) of two naturally occurring enzymes that cannot be evolutionarily related, but which catalyze an identical reaction. (In the classic example of convergent evolution between the trypsin family and subtilisin, the enzymes do not have an identical substrate specificity.) Altman and colleagues propose that a single trivial name be used only for members of a family of homologous macromolecules; in other words, that different trivial names be given to enzymes that catalyze the same precursor–product conversion but do so with different catalytic mechanisms, or which are not members of a single family of homologous macromolecules.

We compared the gene expression patterns of fetal and adult porcine brains and identified a sequence tag that was more abundant in adult than in fetal brain. The RNA corresponding to the sequence tag has the highest expression level in adult cerebellum. Lower expression levels of the transcript were found in adult cerebrum, pituitary, and uterus, as well as in fetal brain, heart, intestine, kidney, and liver. The sequence tag was used to screen a cDNA library from adult porcine brain. Two independent clones of 2,273 nt and 1,701 nt were isolated. The shorter cDNA is a 5′-truncated form of the longer clone, and both clones have almost identical sequences with multiple start and stop codons in all three reading frames. Screening of two different human brain cDNA libraries with porcine cDNA probes resulted in four overlapping cDNA fragments, which were assembled to one contig of 2,336 nt in length. Like noncoding RNAs, the porcine and human sequences have no common conserved open reading frame and share stretches of high homology interrupted by stretches with almost no homology. The human and porcine RNAs were named UM 9(5)h and UM 9(5)p, respectively. They are part of larger transcripts, which are transcribed from single-copy genes, they have very similar tissue distributions, and their sequences are colinear with the respective genomic fragment.

The wild-type transcript of Escherichia coli tRNASec, characterized by a peculiar core architecture and a large variable region, was shown to be aspartylatable by yeast AspRS. Similar activities were found for tRNASec mutants with methionine, leucine, and tryptophan anticodons. The charging efficiency of these molecules was found comparable to that of a minihelix derived from tRNAAsp and is accounted for by the presence of the discriminator residue G73, which is a major aspartate identity determinant. Introducing the aspartate identity elements from the anticodon loop (G34, U35, C36, C38) into tRNASec transforms this molecule into an aspartate acceptor with kinetic properties identical to tRNAAsp. Expression of the aspartate identity set in tRNASec is independent of the size of its variable region. The functional study was completed by footprinting experiments with four different nucleases as structural probes. Protection patterns by AspRS of transplanted tRNASec and tRNAAsp were found similar. They are modified, particularly in the anticodon loop, upon changing the aspartate anticodon into that of methionine. Altogether, it appears that recognition of a tRNA by AspRS is more governed by the presence of the aspartate identity set than by the structural framework that carries this set.