Catalytic complexes of nuclear ribonuclease P (RNase P) ribonucleoproteins are composed of several protein subunits that appear to have specific roles in enzyme function in tRNA processing. This review describes recent progress made in the characterization of human RNase P, its relationship with the ribosomal RNA processing ribonucleoprotein RNase MRP, and the unexpected evolutionary conservation of its subunits. A new model for the biosynthesis of human RNase P is presented, in which this process is dynamic, transcription-dependent, and implicates functionally distinct nuclear compartments in tRNA biogenesis.

The conversion of the single-stranded RNA genome into double-stranded DNA by virus-coded reverse transcriptase (RT) is an essential step of the retrovirus life cycle. In human immunodeficiency virus type 1 (HIV-1), RT uses the cellular tRNA3Lys to initiate the (−) strand DNA synthesis. Placement of the primer tRNA3Lys involves binding of its 3′-terminal 18 nt to a complementary region of genomic RNA termed PBS. However, the PBS sequence is not the unique determinant of primer usage and additional contacts are important. This placement is believed to be achieved in vivo by the nucleocapsid domain of Gag or by the mature protein NCp. Up to now, structural information essentially arose from heat-annealed primer-template complexes (Isel et al., J Mol Biol, 1995, 247:236–250; Isel et al., EMBO J, 1999, 18:1038–1048). Here, we investigated the formation of the primer–template complex mediated by NCp and compared structural and functional properties of heat- and NCp-annealed complexes. We showed that both heat- and NCp-mediated procedures allow comparable high yields of annealing. Then, we investigated structural features of both kinds of complexes by enzymatic probing, and we compared their relative efficiency in (−) strong stop DNA synthesis. We did not find any significant differences between these complexes, suggesting that information derived from the heat-annealed complex can be transposed to the NCp-mediated complex and most likely to complexes formed in vivo.

Translating ribosomes can shift reading frame at specific sites with high efficiency for gene expression purposes. The most common type of shift to the −1 frame involves a tandem realignment of two anticodons from pairing with mRNA sequence of the form X XXY YYZ to XXX YYY Z where the spaces indicate the reading frame. The predominant −1 shift site of this type in eubacteria is A AAA AAG. The present work shows that in Escherichia coli the identity of the 6 nt 3′ of this sequence can be responsible for a 14-fold variation in frameshift frequency. The first 3′ nucleotide has the primary effect, with, in order of decreasing efficiency, U > C > A > G. This effect is independent of other stimulators of frameshifting. It is detected with other X XXA AAG sequences, but not with several other heptameric −1 shift sites. Pairing of E. coli tRNALys with AAG is especially weak at the third codon position. We propose that strong stacking of purines 3′ of AAG stabilizes pairing of tRNALys, diminishing the chance of codon:anticodon dissociation that is a prerequisite for the realignment involved in frameshifting.

Expression of replication-dependent histone genes requires a conserved hairpin RNA element in the 3′ untranslated regions of poly(A)-less histone mRNAs. The 3′ hairpin element is recognized by the hairpin-binding protein or stem-loop-binding protein (HBP/SLBP). This protein–RNA interaction is important for the endonucleolytic cleavage generating the mature mRNA 3′ end. The 3′ hairpin and presumably HBP/SLBP are also required for nucleo-cytoplasmic transport, translation, and stability of histone mRNAs. RNA 3′ processing and mRNA stability are both regulated during the cell cycle. Here, we have determined the three-dimensional structure of a 24-mer RNA comprising a mammalian histone RNA hairpin using heteronuclear multidimensional NMR spectroscopy. The hairpin adopts a novel UUUC tetraloop conformation that is stabilized by base stacking involving the first and third loop uridines and a closing U-A base pair, and by hydrogen bonding between the first and third uridines in the tetraloop. The HBP interaction of hairpin RNA variants was analyzed in band shift experiments. Particularly important interactions for HBP recognition are mediated by the closing U-A base pair and the first and third loop uridines, whose Watson–Crick functional groups are exposed towards the major groove of the RNA hairpin. The results obtained provide novel structural insight into the interaction of the histone 3′ hairpin with HBP, and thus the regulation of histone mRNA metabolism.

The branchpoint sequence and associated polypyrimidine tract are firmly established splicing signals in vertebrates. In plants, however, these signals have not been characterized in detail. The potato invertase mini-exon 2 (9 nt) requires a branchpoint sequence positioned around 50 nt upstream of the 5′ splice site of the neighboring intron and a U11 element found adjacent to the branchpoint in the upstream intron (Simpson et al., RNA, 2000, 6:422–433). Utilizing the sensitivity of this plant splicing system, these elements have been characterized by systematic mutation and analysis of the effect on inclusion of the mini-exon. Mutation of the branchpoint sequence in all possible positions demonstrated that branchpoints matching the consensus, CURAY, were most efficient at supporting splicing. Branchpoint sequences that differed from this consensus were still able to permit mini-exon inclusion but at greatly reduced levels. Mutation of the downstream U11 element suggested that it functioned as a polypyrimidine tract rather than a UA-rich element, common to plant introns. The minimum sequence requirement of the polypyrimidine tract for efficient splicing was two closely positioned groups of uridines 3–4 nt long (<6 nt apart) that, within the context of the mini-exon system, required being close (<14 nt) to the branchpoint sequence. The functional characterization of the branchpoint sequence and polypyrimidine tract defines these sequences in plants for the first time, and firmly establishes polypyrimidine tracts as important signals in splicing of at least some plant introns.

All mitochondrial tRNAs of the kinetoplastid protozoan Leishmania tarentolae are encoded in the nucleus and are imported from the cytosol into the mitochondrion. We previously reported the partitioning of five tRNAs and found that all were shared between the two compartments to different extents. To increase our knowledge of the tRNAs of this organism, and to attempt to understand the signals involved in their subcellular localization, a method to RT-PCR amplify new tRNAs was developed. Various tRNAs were 3′ polyadenylated and reverse transcribed with a sequence-tagged primer. The cDNA was tagged by ligation to an anchor oligonucleotide, and the resulting double-tagged cDNA was amplified by PCR. Four new tRNAs were obtained, bringing to 20 the total number of L. tarentolae tRNAs identified to date. The subcellular localization of 17 tRNAs was quantitatively analyzed by two-dimensional gel electrophoresis and northern hybridization. In general, the previously suggested operational classification of tRNAs into three groups (mainly cytosolic, mainly mitochondrial, and shared between the two compartments) is still valid, but the relative abundance of each tRNA in the cytosol or mitochondrion varied greatly as did the level of expression.

The C-to-U editing of apolipoprotein-B (apo-B) mRNA is catalyzed by an enzyme complex that recognizes an 11-nt mooring sequence downstream of the editing site. A minimal holoenzyme that edits apo-B mRNA in vitro has been defined. This complex contains apobec-1, the catalytic subunit, and apobec-1 complementation factor (ACF), the RNA-binding subunit that binds to the mooring sequence. Here, we show that ACF binds with high affinity to single-stranded but not double-stranded apo-B mRNA. ACF contains three nonidentical RNA recognition motifs (RRM) and a unique C-terminal auxiliary domain. In many multi-RRM proteins, the RRMs mediate RNA binding and an auxiliary domain functions in protein–protein interactions. Here we show that ACF does not fit this simple model. Based on deletion mutagenesis, the RRMs in ACF are necessary but not sufficient for binding to apo-B mRNA. Amino acids in the pre-RRM region are required for complementing activity and RNA binding, but not for interaction with apobec-1. The C-terminal 196 amino acids are not absolutely essential for function. However, further deletion of an RG-rich region from the auxiliary domain abolished complementing activity, RNA binding, and apobec-1 interaction. The auxiliary domain alone did not bind apobec-1. Although all three RRMs are required for complementing activity and apobec-1 interaction, the individual motifs contribute differently to RNA binding. Point mutations in RRM1 or RRM2 decreased the Kd for apo-B mRNA by two orders of magnitude whereas mutations in RRM3 reduced binding affinity 13-fold. The pairwise expression of RRM1 with RRM2 or RRM3 resulted in moderate affinity binding.

The 3′ end of replication-dependent histone mRNAs terminate in a conserved sequence containing a stem-loop. This 26-nt sequence is the binding site for a protein, stem-loop binding protein (SLBP), that is involved in multiple aspects of histone mRNA metabolism and regulation. We have determined the structure of the 26-nt sequence by multidimensional NMR spectroscopy. There is a 16-nt stem-loop motif, with a conserved 6-bp stem and a 4-nt loop. The loop is closed by a conserved U[bull ]A base pair that terminates the canonical A-form stem. The pyrimidine-rich 4-nt loop, UUUC, is well organized with the three uridines stacking on the helix, and the fourth base extending across the major groove into the solvent. The flanking nucleotides at the base of the hairpin stem do not assume a unique conformation, despite the fact that the 5′ flanking nucleotides are a critical component of the SLBP binding site.

Conversion of tRNA precursors to their mature forms requires the action of both endo- and exoribonucleases. Although studies over many years identified the endoribonuclease, RNase P, and several exoribonucleases as the enzymes responsible for generating the mature 5′ and 3′ termini, respectively, of Escherichia coli tRNAs, relatively little is known about how tRNAs are separated from long multimeric or multifunction transcripts, or from long leader and trailer sequences. To examine this question, the tRNA products that accumulate in mutant strains devoid of multiple exoribonucleases plus one or several endoribonucleases were analyzed by northern analysis. We find that the multifunction tyrT transcript, which contains two tRNA1Tyr sequences separated by a 209-nt spacer region plus a downstream mRNA, is cleaved at three sites in the spacer region by the endoribonuclease, RNase E. When both RNase E and RNase P are absent, a product containing both tRNAs accumulates. Two multimeric tRNA transcripts, those for tRNA Arg-His-Leu-Pro and tRNA Gly-Cys-Leu also require RNase E for maturation. For the former transcript, products with long 3′ extensions on tRNAArg, tRNAHis, and tRNAPro, as well as the primary transcript, accumulate in the absence of RNase E. For the latter transcript, RNase E cleaves downstream of each tRNA. Little processing of either multimeric transcript occurs in the absence of both RNase E and RNase P. These data indicate that RNase E is a major contributor to the initial processing of E. coli tRNA transcripts, providing substrates for final maturation by RNase P and the 3′ exoribonucleases. Based on this new information, a detailed model for tRNA maturation is proposed.

In the Bromoviridae family of plant viruses, trinucleotide hairpin loops play an important role in RNA transcription. Recently, we reported that Brome mosaic virus (BMV) subgenomic (sg) transcription depended on the formation of an unusual triloop hairpin. By native gel electrophoresis, enzymatic structure probing, and NMR spectroscopy it is shown here that in the absence of viral replicase the hexanucleotide loop 5′C1AUAG5A3′ of this RNA structure can adopt a pseudo trinucleotide loop conformation by transloop base pairing between C1 and G5. By means of in vitro replication assays using partially purified BMV RNA-dependent RNA polymerase (RdRp) it was found that other base pairs contribute to sg transcription, probably by stabilizing the formation of this pseudo triloop, which is proposed to be the primary element recognized by the viral replicase. The BMV pseudo triloop structure strongly resembles iron-responsive elements (IREs) in cellular messenger RNAs and may represent a general protein-binding motif. In addition, in vitro replication assays showed that the BMV sg hairpin is functionally equivalent to the minus-strand core promoter hairpin stem-loop C at the 3′ end of BMV RNAs. Replacement of the sg hairpin by stem-loop C yielded increased sg promoter activity whereas replacement of stem-loop C by the sg hairpin resulted in reduced minus-strand promoter activity. We conclude that AUA triloops represent the common motif in the BMV sg and minus-strand promoters required for recruitment of the viral replicase. Additional sequence elements of the minus-strand promoter are proposed to direct the RdRp to the initiation site at the 3′ end of the genomic RNA.