lsm1 mutations impairing the ability of the Lsm1p-7p-Pat1p complex to preferentially bind to oligoadenylated RNA affect mRNA decay in vivo

The poly(A) tail is a crucial determinant in the control of both mRNA translation and decay. Poly(A) tail length dictates the triggering of the degradation of the message body in the major 5′ to 3′ and 3′ to 5′ mRNA decay pathways of eukaryotes. In the 5′ to 3′ pathway oligoadenylated but not polyadenylated mRNAs are selectively decapped in vivo, allowing their subsequent degradation by 5′ to 3′ exonucleolysis. The conserved Lsm1p-7p-Pat1p complex is required for normal rates of decapping in vivo, and the purified complex exhibits strong binding preference for oligoadenylated RNAs over polyadenylated or unadenylated RNAs in vitro. In the present study, we show that two lsm1 mutants produce mutant complexes that fail to exhibit such higher affinity for oligoadenylated RNA in vitro. Interestingly, these mutant complexes are normal with regard to their integrity and retain the characteristic RNA binding properties of the wild-type complex, namely, binding near the 3′-end of the RNA, having higher affinity for unadenylated RNAs that carry U-tracts near the 3′-end over those that do not and exhibiting similar affinities for unadenylated and polyadenylated RNAs. Yet, these lsm1 mutants exhibit a strong mRNA decay defect in vivo. These results underscore the importance of Lsm1p-7p-Pat1p complex–mRNA interaction for mRNA decay in vivo and imply that the oligo(A) tail mediated enhancement of such interaction is crucial in that process.     

The RNA ligands for mouse proline-rich RNA-binding protein (mouse Prrp) contain two consensus sequences in separate loop structure

Mouse proline-rich RNA-binding protein (mPrrp) is a mouse ortholog of Xenopus Prrp, which binds to a vegetal localization element (VLE) in the 3′-untranslated region (3′-UTR) of Vg1 mRNA and is expected to be involved in the transport and/or localization of Vg1 mRNA to the vegetal cortex of oocytes. In mouse testis, mPrrp protein is abundantly expressed in the nuclei of pachytene spermatocytes and round spermatids, and shifts to the cytoplasm in elongating spermatids. To gain an insight into the function of mPrrp in male germ cells, we performed in vitro RNA selection (SELEX) to determine the RNA ligand sequence of mPrrp. This analysis revealed that many of the selected clones contained both of two conserved elements, AAAUAG and GU_1–3AG. RNA-binding study on deletion mutants and secondary structure analyses of the selected RNA revealed that a two-loop structure containing the conserved elements is required for high-affinity binding to mPrrp. Furthermore, we found that the target mRNAs of Xenopus Prrp contain intact AAAUAG and GU_1–3AG sequences in the 3′-UTR, suggesting that these binding sequences are shared by Prrps of Xenopus and mouse.     

RNAi-mediated depletion of the 15 KH domain protein, vigilin, induces death of dividing and non-dividing human cells but does not initially inhibit protein synthesis

Vigilin/Scp160p/DDP1 is a ubiquitous and highly conserved protein containing 15 related, but non-identical, K-homology (KH) nucleic acid binding domains. While its precise function remains unknown, proposed roles for vigilin include chromosome partitioning at mitosis, facilitating translation and tRNA transport, and control of mRNA metabolism, including estrogen-mediated stabilization of vitellogenin mRNA. To probe sites of vigilin action in vertebrate cells, we performed nucleic acid binding and RNA interference studies. Consistent with a potential role in chromosome partitioning, human vigilin exhibits a higher affinity for Drosophila dodecasatellite single-stranded DNA than for vitellogenin mRNA 3′-UTR. Direct observation and flow cytometry in non-mitotic, serum-starved, HeLa cells showed that RNAi-mediated vigilin knockdown is rapidly lethal, indicating an essential function for vigilin distinct from its proposed role in chromosome partitioning. Pulse labeling experiments revealed that rates of protein synthesis and degradation are unaffected by the several fold reduction in vigilin levels early in siRNA knockdown indicating that vigilin is not a global regulator of translation. These data show that vigilin is an essential protein in human cells, support the view that vigilin’s most essential functions are neither chromosome partitioning nor control of translation, and are consistent with vigilin playing a critical role in cytoplasmic mRNA metabolism.     

Structural insights into the targeting of mRNA GU-rich elements by the three RRMs of CELF1

The CUG-BP, Elav-like family (CELF) of RNA-binding proteins control gene expression at a number of different levels by regulating pre-mRNA splicing, deadenylation and mRNA stability. We present structural insights into the binding selectivity of CELF member 1 (CELF1) for GU-rich mRNA target sequences of the general form 5′-UGUN_xUGUN_yUGU and identify a high affinity interaction (K_d ∼ 100 nM for x = 2 and y = 4) with simultaneous binding of all three RNA recognition motifs within a single 15-nt binding element. RNA substrates spin-labelled at either the 3′ or 5′ terminus result in differential nuclear magnetic resonance paramagnetic relaxation enhancement effects, which are consistent with a non-sequential 2-1-3 arrangement of the three RNA recognition motifs on UGU sites in a 5′ to 3′ orientation along the RNA target. We further demonstrate that CELF1 binds to dispersed single-stranded UGU sites at the base of an RNA hairpin providing a structural rationale for recognition of CUG expansion repeats and splice site junctions in the regulation of alternative splicing.     

The Putative Nucleic Acid Helicase Sen1p Is Required for Formation and Stability of Termini and for Maximal Rates of Synthesis and Levels of Accumulation of Small Nucleolar RNAs in Saccharomyces cerevisiae

Sen1p from Saccharomyces cerevisiae is a nucleic acid helicase related to DEAD box RNA helicases and type I DNA helicases. The temperature-sensitive sen1-1 mutation located in the helicase motif alters the accumulation of pre-tRNAs, pre-rRNAs, and some small nuclear RNAs. In this report, we show that cells carrying sen1-1 exhibit altered accumulation of several small nucleolar RNAs (snoRNAs) immediately upon temperature shift. Using Northern blotting, RNase H cleavage, primer extension, and base compositional analysis, we detected three forms of the snoRNA snR13 in wild-type cells: an abundant TMG-capped 124-nucleotide (nt) mature form (snR13F) and two less abundant RNAs, including a heterogeneous population of ~1,400-nt 3′-extended forms (snR13R) and a 108-nt 5′-truncated form (snR13T) that is missing 16 nt at the 5′ end. A subpopulation of snR13R contains the same 5′ truncation. Newly synthesized snR13R RNA accumulates with time at the expense of snR13F following temperature shift of sen1-1 cells, suggesting a possible precursor-product relationship. snR13R and snR13T both increase in abundance at the restrictive temperature, indicating that Sen1p stabilizes the 5′ end and promotes maturation of the 3′ end. snR13F contains canonical C and D boxes common to many snoRNAs. The 5′ end of snR13T and the 3′ end of snR13F reside within C₂U₄ sequences that immediately flank the C and D boxes. A mutation in the 5′ C₂U₄ repeat causes underaccumulation of snR13F, whereas mutations in the 3′ C₂U₄ repeat cause the accumulation of two novel RNAs that migrate in the 500-nt range. At the restrictive temperature, double mutants carrying sen1-1 and mutations in the 3′ C₂U₄ repeat show reduced accumulation of the novel RNAs and increased accumulation of snR13R RNA, indicating that Sen1p and the 3′ C₂U₄ sequence act in a common pathway to facilitate 3′ end formation. Based on these findings, we propose that Sen1p and the C₂U₄ repeats that flank the C and D boxes promote maturation of the 3′ terminus and stability of the 5′ terminus and are required for maximal rates of synthesis and levels of accumulation of mature snR13F.     

Rotavirus RNA Replication Requires a Single-Stranded 3′ End for Efficient Minus-Strand Synthesis

The segmented double-stranded (ds) RNA genome of the rotaviruses is replicated asymmetrically, with viral mRNA serving as the template for the synthesis of minus-strand RNA. Previous studies with cell-free replication systems have shown that the highly conserved termini of rotavirus gene 8 and 9 mRNAs contain cis-acting signals that promote the synthesis of dsRNA. Based on the location of the cis-acting signals and computer modeling of their secondary structure, the ends of the gene 8 or 9 mRNAs are proposed to interact in cis to form a modified panhandle structure that promotes the synthesis of dsRNA. In this structure, the last 11 to 12 nucleotides of the RNA, including the cis-acting signal that is essential for RNA replication, extend as a single-stranded tail from the panhandled region, and the 5′ untranslated region folds to form a stem-loop motif. To understand the importance of the predicted secondary structure in minus-strand synthesis, mutations were introduced into viral RNAs which affected the 3′ tail and the 5′ stem-loop. Analysis of the RNAs with a cell-free replication system showed that, in contrast to mutations which altered the structure of the 5′ stem-loop, mutations which caused complete or near-complete complementarity between the 5′ end and the 3′ tail significantly inhibited (≥10-fold) minus-strand synthesis. Likewise, incubation of wild-type RNAs with oligonucleotides which were complementary to the 3′ tail inhibited replication. Despite their replication-defective phenotype, mutant RNAs with complementary 5′ and 3′ termini were shown to competitively interfere with the replication of wild-type mRNA and to bind the viral RNA polymerase VP1 as efficiently as wild-type RNA. These results indicate that the single-strand nature of the 3′ end of rotavirus mRNA is essential for efficient dsRNA synthesis and that the specific binding of the RNA polymerase to the mRNA template is required but not sufficient for the synthesis of minus-strand RNA.     

The rpoS mRNA leader recruits Hfq to facilitate annealing with DsrA sRNA

Small noncoding RNAs (sRNAs) regulate the response of bacteria to environmental stress in conjunction with the Sm-like RNA binding protein Hfq. DsrA sRNA stimulates translation of the RpoS stress response factor in Escherichia coli by base-pairing with the 5′ leader of the rpoS mRNA and opening a stem–loop that represses translation initiation. We report that rpoS leader sequences upstream of this stem–loop greatly increase the sensitivity of rpoS mRNA to Hfq and DsrA. Native gel mobility shift assays show that Hfq increases the rate of DsrA binding to the full 576 nt rpoS leader as much as 50-fold. By contrast, base-pairing with a 138-nt RNA containing just the repressor stem–loop is accelerated only twofold. Deletion and mutagenesis experiments showed that sensitivity to Hfq requires an upstream AAYAA sequence. Leaders long enough to contain this sequence bind Hfq tightly and form stable ternary complexes with Hfq and DsrA. A model is proposed in which Hfq recruits DsrA to the rpoS mRNA by binding both RNAs, releasing the self-repressing structure in the mRNA. Once base-pairing between DsrA and rpoS mRNA is established, interactions between Hfq and the mRNA may stabilize the RNA complex by removing Hfq from the sRNA.     

Characterization of Influenza Virus PB1 Protein Binding to Viral RNA: Two Separate Regions of the Protein Contribute to the Interaction Domain

The interaction of the PB1 subunit of the influenza virus polymerase with the viral RNA (vRNA) template has been studied in vitro. The experimental approach included the in vitro binding of labeled model vRNA to PB1 protein immobilized as an immunoprecipitate, as well as Northwestern analyses. The binding to model vRNA was specific, and an apparent K_d of about 2 × 10⁻⁸ M was determined. Although interaction with the isolated 3′ arm of the panhandle was detectable, interaction with the 5′ arm was prominent and the binding was optimal with a panhandle analog structure (5′+3′ probe). When presented with a panhandle analog mixed probe, PB1 was able to retain the 3′ arm as efficiently as the 5′ arm. The sequences of the PB1 protein involved in vRNA binding were identified by in vitro interaction tests with PB1 deletion mutants. Two separate regions of the PB1 protein sequence proved positive for binding: the N-terminal 83 amino acids and the C-proximal sequences located downstream of position 493. All mutants able to interact with model vRNA were capable of binding the 5′ arm more efficiently than the 3′ arm of the panhandle. Taken together, these results suggest that two separate regions of the PB1 protein constitute a vRNA binding site that interacts preferentially with the 5′ arm of the panhandle structure.     

Regulation of small RNA stability: methylation and beyond

As central components of RNA silencing, small RNAs play diverse and important roles in many biological processes in eukaryotes. Aberrant reduction or elevation in the levels of small RNAs is associated with many developmental and physiological defects. The in vivo levels of small RNAs are precisely regulated through modulating the rates of their biogenesis and turnover. 2′-O-methylation on the 3′ terminal ribose is a major mechanism that increases the stability of small RNAs. The small RNA methyltransferase HUA ENHANCER1 (HEN1) and its homologs methylate microRNAs and small interfering RNAs (siRNAs) in plants, Piwi-interacting RNAs (piRNAs) in animals, and siRNAs in Drosophila. 3′ nucleotide addition, especially uridylation, and 3′-5′ exonucleolytic degradation are major mechanisms that turnover small RNAs. Other mechanisms impacting small RNA stability include complementary RNAs, cis-elements in small RNA sequences and RNA-binding proteins. Investigations are ongoing to further understand how small RNA stability impacts their accumulation in vivo in order to improve the utilization of RNA silencing in biotechnology and therapeutic applications.     

Two-dimensional combinatorial screening and the RNA Privileged Space Predictor program efficiently identify aminoglycoside–RNA hairpin loop interactions

Herein, we report the identification of RNA hairpin loops that bind derivatives of kanamycin A, tobramycin, neamine, and neomycin B via two-dimensional combinatorial screening, a method that screens chemical and RNA spaces simultaneously. An arrayed aminoglycoside library was probed for binding to a 6-nucleotide RNA hairpin loop library (4096 members). Members of the loop library that bound each aminoglycoside were excised from the array, amplified and sequenced. Sequences were analyzed with our newly developed RNA Privileged Space Predictor (RNA-PSP) program, which analyzes selected sequences to identify statistically significant trends. RNA-PSP identified the following unique trends: 5′UNNNC3′ loops for the kanamycin A derivative (where N is any nucleotide); 5′UNNC3′ loops for the tobramycin derivative; 5′UNC3′ loops for the neamine derivative; and 5′UNNG3′ loops for the neomycin B derivative. The affinities and selectivities of a subset of the ligand–hairpin loop interactions were determined. The selected interactions have K_d values ranging from 10 nM to 605 nM. Selectivities ranged from 0.4 to >200-fold. Interestingly, the results from RNA-PSP are able to qualitatively predict specificity based on overlap between the RNA sequences selected for the ligands. These studies expand the information available on small molecule–RNA motif interactions, which could be useful to design ligands targeting RNA.     

Mutational and Structural Analysis of the Tandem Zinc Finger Domain of Tristetraprolin

Tristetraprolin (TTP), the best known member of a class of tandem (R/K)YKTELCX₈CX₅CX₃H zinc finger proteins, can destabilize target mRNAs by first binding to AU-rich elements (AREs) in their 3′-untranslated regions (UTRs) and subsequently promoting deadenylation and ultimate destruction of those mRNAs. This study sought to determine the roles of selected amino acids in the RNA binding domain, known as the tandem zinc finger (TZF) domain, in the ability of the full-length protein to bind to AREs within the tumor necrosis factor α (TNF) mRNA 3′-UTR. Within the CX₈C region of the TZF domain, mutation of some of the residues specific to TTP, not found in other members of the TTP protein family, resulted in decreased binding to RNA as well as inhibited mRNA deadenylation and decay. Evaluation of simulation solution models revealed a distinct structure in the second zinc finger of TTP that was induced by the presence of these TTP-specific residues. In addition, mutations within the lead-in sequences preceding the first C of highly conserved residues within the CX₅C or CX₃H regions or within the linker region between the two fingers also perturbed both RNA binding and the simulation model of the TZF domain in complex with RNA. We conclude that, although the majority of conserved residues within the TZF domain of TTP are required for productive binding, not all residues at sequence-equivalent positions in the two zinc fingers of the TZF domain of TTP are functionally equivalent.     

The 3' untranslated region of human vimentin mRNA interacts with protein complexes containing eEF-1gamma and HAX-1

Previously, we have shown that the vimentin 3′ untranslated region (3′UTR) contains a highly conserved region, which is sufficient for the perinuclear localization of a reporter mRNA. This region was shown to specifically bind protein(s) by band shift analyses. UV-cross-linking studies suggest these proteins are 46- and 35-kDa in mass. Here, we have used this sequence as ‘bait’ to isolate RNA binding proteins using the yeast three-hybrid method. This technique relies on a functional assay detecting bona fide RNA–protein interaction in vivo. Three cDNA isolates, HAX-1, eEF-1γ and hRIP, code for proteins of a size consistent with in vitro cross- linking studies. In all cases, recombinant proteins were capable of binding RNA in vitro. Although hRIP is thought to be a general mRNA binding protein, this represents an unreported activity for eEF-1γ and HAX-1. Moreover, HAX-1 binding appears to be specific to vimentin’s 3′UTR. Both in vivo synthesized eEF-1γ and HAX-1 proteins were ‘pulled out’ of HeLa whole cell extracts by binding to a RNA affinity column comprised of vimentin’s 3′UTR. Moreover, size-fractionation of extracts results in the separation of large complexes containing either eEF-1γ or HAX-1. Thus, in addition to their known functions, both eEF-1γ and HAX-1 are RNA binding proteins, which suggests new roles in mRNA translation and/or perinuclear localization.     

Messenger RNA processing in Methanocaldococcus (Methanococcus) jannaschii

Messenger RNA (mRNA) processing plays important roles in gene expression in all domains of life. A number of cases of mRNA cleavage have been documented in Archaea, but available data are fragmentary. We have examined RNAs present in Methanocaldococcus (Methanococcus) jannaschii for evidence of RNA processing upstream of protein-coding genes. Of 123 regions covered by the data, 31 were found to be processed, with 30 including a cleavage site 12–16 nucleotides upstream of the corresponding translation start site. Analyses with 3′-RACE (rapid amplification of cDNA ends) and 5′-RACE indicate that the processing is endonucleolytic. Analyses of the sequences surrounding the processing sites for functional sites, sequence motifs, or potential RNA secondary structure elements did not reveal any recurring features except for an AUG translation start codon and (in most cases) a ribosome binding site. These properties differ from those of all previously described mRNA processing systems. Our data suggest that the processing alters the representation of various genes in the RNA pool and therefore, may play a significant role in defining the balance of proteins in the cell.     

Enzymatic characterization of mRNA cap adenosine-N6 methyltransferase PCIF1 activity on uncapped RNAs

The phosphorylated RNA polymerase II CTD interacting factor 1 (PCIF1) is a methyltransferase that adds a methyl group to the N6-position of 2′O-methyladenosine (A_m), generating N6, 2′O-dimethyladenosine (m⁶A_m) when A_m is the cap-proximal nucleotide. In addition, PCIF1 has ancillary methylation activities on internal adenosines (both A and A_m), although with much lower catalytic efficiency relative to that of its preferred cap substrate. The PCIF1 preference for 2′O-methylated A_m over unmodified A nucleosides is due mainly to increased binding affinity for A_m. Importantly, it was recently reported that PCIF1 can methylate viral RNA. Although some viral RNA can be translated in the absence of a cap, it is unclear what roles PCIF1 modifications may play in the functionality of viral RNAs. Here we show, using in vitro assays of binding and methyltransfer, that PCIF1 binds an uncapped 5′-A_m oligonucleotide with approximately the same affinity as that of a cap analog (K_M = 0.4 versus 0.3 μM). In addition, PCIF1 methylates the uncapped 5′-A_m with activity decreased by only fivefold to sixfold compared with its preferred capped substrate. We finally discuss the relationship between PCIF1-catalyzed RNA methylation, shown here to have broader substrate specificity than previously appreciated, and that of the RNA demethylase fat mass and obesity-associated protein (FTO), which demonstrates PCIF1-opposing activities on capped RNAs.     

Masked antisense: a molecular configuration for discriminating similar RNA targets

Antisense technology has great potential for the control of RNA expression, but there remain few successful applications of the technology. Expressed antisense RNA can effectively down-regulate expression of a gene over long periods, but cannot differentiate partly identical sequences, such as the mRNA of fusion genes or those with point mutants. We have designed a structured form of expressed antisense, which can discriminate between highly similar mRNA molecules. These ‘masked’ antisense RNAs have most of the antisense sequence sequestered within duplex elements, leaving a short single-stranded region to initiate binding to target RNA. After contacting the correct target, the structured RNA can unravel, releasing the masked antisense region to form a stable duplex with the mRNA. We demonstrate that suitable masked antisense RNA can discriminate between the two forms of BCR–ABL mRNA that result from the Philadelphia chromosomal translocations, as well as discriminating the normal BCR and ABL mRNA.