Ram Reddy

Upstream regulatory elements are necessary and sufficient for transcription of a U6 RNA gene by RNA polymerase III.

Whereas the genes coding for trimethyl guanosine‐capped snRNAs are transcribed by RNA polymerase II, the U6 RNA genes are transcribed by RNA polymerase III. In this study, we have analyzed the cis‐regulatory elements involved in the transcription of a mouse U6 snRNA gene in vitro and in frog oocytes. Transcriptional analysis of mutant U6 gene constructs showed that, unlike most known cases of polymerase III transcription, intragenic sequences except the initiation nucleotide are dispensable for efficient and accurate transcription of U6 gene in vitro. Transcription of 5′ deletion mutants in vitro and in frog oocytes showed that the upstream region, within 79 bp from the initiation nucleotide, contains elements necessary for U6 gene transcription. Transcription studies were carried out in frog oocytes with U6 genes containing 5′ distal sequence; these studies revealed that the distal element acts as an orientation‐dependent enhancer when present upstream to the gene, while it is orientation‐independent but distance‐dependent enhancer when placed down‐stream to the U6 gene. Analysis of 3′ deletion mutants showed that the transcription termination of U6 RNA is dependent on a T cluster present on the 3′ end of the gene, thus providing further support to other lines of evidence that U6 genes are transcribed by RNA polymerase III. These observations suggest the involvement of a composite of components of RNA polymerase II and III transcription machineries in the transcription of U6 genes by RNA polymerase III.

p110, a novel human U6 snRNP protein and U4/U6 snRNP recycling factor

During each spliceosome cycle, the U6 snRNA undergoes extensive structural rearrangements, alternating between singular, U4–U6 and U6–U2 base‐paired forms. In Saccharomyces cerevisiae, Prp24 functions as an snRNP recycling factor, reannealing U4 and U6 snRNAs. By database searching, we have identified a Prp24‐related human protein previously described as p110nrb or SART3. p110 contains in its C‐terminal region two RNA recognition motifs (RRMs). The N‐terminal two‐thirds of p110, for which there is no counterpart in the S.cerevisiae Prp24, carries seven tetratricopeptide repeat (TPR) domains. p110 homologs sharing the same domain structure also exist in several other eukaryotes. p110 is associated with the mammalian U6 and U4/U6 snRNPs, but not with U4/U5/U6 tri‐snRNPs nor with spliceosomes. Recombinant p110 binds in vitro specifically to human U6 snRNA, requiring an internal U6 region. Using an in vitro recycling assay, we demonstrate that p110 functions in the reassembly of the U4/U6 snRNP. In summary, p110 represents the human ortholog of Prp24, and associates only transiently with U6 and U4/U6 snRNPs during the recycling phase of the spliceosome cycle.

The 40-kilodalton to autoantigen associates with nucleotides 21 to 64 of human mitochondrial RNA processing/7-2 RNA in vitro.

A 40-kDa To antigen recognized by sera from some patients with autoimmune diseases is an integral component of both human RNase P and mitochondrial RNA processing (MRP) RNase. Human MRP and RNase P RNAs, synthesized in vitro, readily associate with the To antigen present in the HeLa cell extract. Using this in vitro reconstitution system, the binding site of the To antigen is localized to a 44-nucleotide-long sequence corresponding to nucleotides 21 to 64 of the human MRP RNA. UV cross-linking experiments showed that the To antigen binds directly to MRP RNA and to RNase P (H1) RNA through RNA-protein interactions. Although the MRP RNA and RNAse P (H1) RNA show sequence homology in four conserved blocks (H. A. Gold, J. N. Topper, D. A. Clayton, and J. Craft, Science 245:1377-1380, 1989), the To antigen-binding site in MRP RNA does not show any obvious primary sequence homology with H1 RNA. These data suggest that the To antigen binds to a conserved and presumably a common secondary or tertiary structure in human MRP and RNase P RNAs.

Small Nuclear RNAs and RNA Processing

I. Summary Ribosomal RNA, transfer RNA, and messenger RNAs, which comprise about 99% of the cellular RNA, are part of the protein-synthesizing machinery. Many studies in the last 15 years have established the presence of another class of RNA, “small nuclear RNAs” (snRNAs) that account for 0.1-1% of the total cellular RNA. There is evidence for at least 15 distinct small RNAs in rat and human cells. Of these, six (designated Ul- to U6-RNAs) are capped, metabolically stable, synthesized by polymerase II, present as ribonucleoprotein particles, and present in concentrations comparable to that of ribosomes. U3-RNA, found only in the nucleolus, is associated with preribosomal RNA and is involved in maturation of ribosomal RNAs, although the precise mechanism is not known. U1-, U2-, U4-, U5-, and U6-RNPs, found in nucleoplasm, are in part associated with hnRNP particles and are implicated in messenger RNA transport and processing; the detailed mechanism(s) are under study. The discovery that patients with autoimmune diseases produce antibodies against RNP particles containing small nuclear RNAs made improved methods for studies on small RNPs available to researchers in many disciplines. The availability of these immunological reagents for selective immunoprecipitation of specific U-snRNPs and other snRNPs offers a powerful approach to study their structures, intracellular localization, and function. In addition, the attractive hypothesis that snRNAs, like Ul-RNA, may be involved in properly aligning splice junctions, of pre-mRNA, brought snRNAs to the attention of many researchers. Unlike the capped U-snRNAs, which are transcribed by RNA polymerase II, the noncapped RNAs are transcribed by RNA polymerase III; they have diverse functions. Cytoplasmic 7 S RNA is an integral part of the “signal recognition particle” involved in synthesis and transport of secretory proteins (181a). RNase-P (EC 3.1.26.5) RNA has been reported (200, 201 ) to be part of an RNP particle involved in processing precursor tRNAs. P-snRNA is implicated in making chromatin accessible for crossing-over during meiosis (34) , and CEH-RNA is thought to induce embryonic heart-cell differentiation (198). In addition, several RNAs, including 4.5 and 4.5 SI, 6 S, 7 S RNAs, exhibit homologies to reiterated DNA sequences, whose significance is not understood. Although the structures of the small RNAs are now well defined, much remains to be learned about their relationship to proteins in snRNP particles and the functions of the snRNP particles.

Methylated cap structures in eukaryotic RNAs: Structure, synthesis and functions

There are more than twenty capped small nuclear RNAs characterized in eukaryotic cells. All the capped RNAs appear to be involved in the processing of other nuclear premessenger or preribosomal RNAs. These RNAs contain either trimethylguanosine (TMG) cap structure or methylated gamma phosphate (Mppp) cap structure. The TMG capped RNAs are capped with M7G during transcription by RNA polymerase II and trimethylated further post-transcriptionally. The Mppp-capped RNAs are transcribed by RNA polymerase III and also capped post-transcriptionally. The cap structures improve the stability of the RNAs and in some cases TMG cap is required for transport of the ribonucleoproteins from cytoplasm to the nucleus. Where tested, the cap structures were not essential for their function in processing other RNAs.

Capping of mammalian U6 small nuclear RNA in vitro is directed by a conserved stem-loop and AUAUAC sequence: conversion of a noncapped RNA into a capped RNA.

The cap structure of U6 small nuclear RNA (snRNA) is gamma-monomethyl phosphate and is distinct from other known RNA cap structures (R. Singh and R. Reddy, Proc. Natl. Acad. Sci. USA 86:8280-8283, 1989). Here we show that the information for capping the U6 snRNA in vitro is within the initial 25 nucleotides of the U6 RNA. The capping determinant in mammalian U6 snRNA is a bipartite element--a phylogenetically conserved stem-loop structure and an AUAUAC sequence, or a part thereof, following this stem-loop. Wild-type capping efficiency was obtained when the AUAUAC motif immediately followed the stem-loop and when the gamma-phosphate of the initiation nucleotide was in close proximity to the capping determinant. Incorporation of a synthetic stem-loop followed by an AUAUAC sequence is sufficient to covert a noncapped heterologous transcript into a capped transcript. Transcripts with the initial 32 nucleotides of Saccharomyces cerevisiae U6 snRNA are accurately capped in HeLa cell extract, indicating that capping machinery from HeLa cells can cap U6 snRNA from an evolutionarily distant eucaryote. The U6-snRNA-specific capping is unusual in that it is RNA sequence dependent, while the capping of mRNAs and other U snRNAs is tightly coupled to transcription and is independent of the RNA sequence.

Formation of 2',3'-cyclic phosphates at the 3' end of human U6 small nuclear RNA in vitro. Identification of 2',3'-cyclic phosphates at the 3' ends of human signal recognition particle and mitochondrial RNA processing RNAs.

Approximately 90% of human U6 small nuclear RNA (snRNA) contains uridine cyclic phosphate (U>p) at its 3′-end (Lund, E., and Dahlberg, J. E. (1992) Science 255, 327–330). We studied the formation of U>p at the 3′ end of human U6 snRNA using an in vitro system where uridylic acid residues are added from UTP precursor and U>p is formed. Analysis of U6 snRNAs with varying number of uridylic acid residues showed that each of these species contains U>p where the phosphate originated from α-phosphate of UTP precursor. The cyclic phosphate formation occurred on U6 snRNA in extracts where essential spliceosomal snRNAs were specifically degraded, thereby indicating that U>p formation is not coupled to pre-mRNA splicing. A subpopulation of human signal recognition particle and mitochondrial RNA processing RNAs isolated from HeLa cells also contained cyclic phosphates at their 3′ ends. These data suggest that U>p in U6 snRNA is unlikely to be related to its participation in splicing of pre-mRNAs. It appears that cyclic phosphate is an intermediate product in the metabolism of these small RNAs.

Small RNA Database

The small RNA database is a compilation of all the small size RNA sequences available to date, including nuclear, nucleolar, cytoplasmic and mitochondria small RNAs from eukaryotic organisms and small RNAs from prokaryotic cells as well as viruses. Currently, approximately 600 small RNA sequences are in our database. It also gives the sources of individual RNAs and their GenBank accession numbers. The small RNA database can be accessed through the WWW (World Wide Web). Our WWW URL address is: http://mbcr.bcm.tmc. edu/smallRNA/smallrna.html . The new small RNA sequences published since our last compilation are listed in this paper (Table 1).

Structural and functional similarities between MRP and RNase P

RNase P, the enzyme responsible for 5′-end processing of tRNAs and 4.5S RNA, has been extensively characterized fromE. coli. The RNA component ofE. coli RNase P, without the protein, has the enzymatic activity and is the first true RNA enzyme to be characterized. RNase P and MRP are two distinct nuclear ribonucleoprotein (RNP) particles characterized in many eukaryotic cells including human, yeast and plant cells. There are many similarities between RNase P and MRP. These include: (1) sequence specific endonuclease activity; (2) homology at the primary and secondary structure levels; and (3) common proteins in both the RNPs. It is likely that RNase P and MRP originated from a common ancestor.

Isolation and characterization of a new 110 kDa human nuclear RNA-binding protein (p110nrb)

RNA-protein interactions play key roles in many fundamental cellular processes such as RNA processing, RNA transport, and RNA translation. During our attempts to isolate the human U6 small nuclear RNA capping enzyme, we identified a new 110 kDa nuclear RNA-binding protein, designated p110nrb. The full-length cDNA clone for p110nrb was characterized, and it encodes a 963 amino acid polypeptide. It is a highly acidic protein (pI 5.28) and the carboxyl terminal portion contains two conserved RNP motifs. A databank search found a putative C. elegans protein that might be the p110nrb homologue. The p110nrb was overexpressed as a glutathione S-transferase fusion protein in insect Sf9 cells, purified by affinity chromatography and injected into rabbits to produce specific polyclonal antibodies. Immunofluorescent staining showed that p110nrb is distributed evenly throughout the nucleoplasm. Northern blots showed that the mRNA is expressed in all tissues examined. An in vitro RNA-binding assay showed that p110nrb bound to RNA. These data suggest that p110nrb may play a role in the metabolism of nuclear RNA.