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RNA Machines of the Mammalian Cell Nucleus

Research Summary

Joan Steitz is interested in the multiple roles played by noncoding RNA–protein complexes in gene expression in vertebrate cells.

Three distinct kinds of RNA molecules orchestrate gene expression in all cells. Messenger RNAs (mRNAs) bring the information from the genome to the protein synthesis factory, the ribosome, which itself is composed mostly of RNA (rRNA). Transfer RNAs (tRNAs) serve as the adapter molecules that align the correct amino acids on the mRNA template during the elongation of protein chains.

A fourth class of RNA molecules, called noncoding RNAs, is also crucial for gene expression in higher cells. Small RNAs (having chain lengths of 60–300 nucleotides), which were the first to be characterized, are highly conserved and particularly abundant in the nuclei of mammalian cells. Small nuclear RNAs (snRNAs) exist tightly bound to one or more proteins in particles called small nuclear ribonucleoproteins (snRNPs: pronounced "snurps"). Some snRNPs inhabit the nucleoplasm, which contains the DNA and is devoted to the production of mRNAs for export to the cytoplasm. Others occupy the nucleolus, where ribosomes are assembled before being shunted to the cytoplasm to function in protein synthesis. More recently, a novel family of tiny regulatory RNAs, dubbed microRNAs because they are only about 22 nucleotides long, has been discovered. They too bind proteins (creating microRNPs), but control gene expression at the level of translation and mRNA stability in the cytoplasm. MicroRNAs play important roles in cancer and infectious disease, as well as in normal development.

Figure 1: The ENE confers stability...

The most numerous snRNPs of the nucleoplasm (the U1, U2, U5, and U4/U6 particles) participate in pre-mRNA splicing, an early step in gene expression. The DNA and pre-mRNAs in mammalian and other higher cells contain nonsense segments called introns, which must be excised for the mRNA to encode a meaningful protein product. In the nucleus, the pre-mRNA is cut, and the sense segments—exons—are precisely joined back together. Splicing occurs in spliceosomes, which are assembled on each intron and include the above snRNPs and a host of protein factors. A much less abundant minor spliceosome removes 1/300 introns from human pre-mRNAs by recognizing slightly different sequences near the ends of these introns. Our studies of the splicing of the minor introns have revealed the participation of one common snRNP (U5) and four snRNPs (U11, U12, U4atac, and U6atac) that are distinct but structurally and functionally analogous to their counterparts in the major spliceosome. Another snRNP related to the spliceosomal snRNPs directs the maturation of histone mRNAs, which are cleaved from longer precursors but not polyadenylated at their 3′ ends (as are the vast majority of vertebrate mRNAs). We have shown that many of the same components contribute to the nuclear maturation of mRNAs both possessing and lacking poly(A) tails, as well as to developmentally controlled poly(A) tail elongation in the cytoplasm, which serves to activate translation.

The nucleus contains additional kinds of snRNPs that contribute to the biogenesis of other classes of RNA. In the nucleolus there are about 200 different small nucleolar RNAs (snoRNAs), packaged with specific proteins to form nucleolar snRNPs (snoRNPs). These participate not only in the cutting of the long pre-rRNA precursor into the mature 18S, 5.8S, and 28S rRNAs but also in rRNA modification. About 100 2′-O-methyl groups and about 100 pseudouridines are introduced at highly conserved rRNA positions by base-pairing between a guide snoRNA and the pre-rRNA. Another group of guide RNAs acts on the snRNAs of the spliceosome, which contain multiple 2′-O-methyl and pseudouridine modifications concentrated in regions that engage in RNA-RNA interactions critical for splicing. Some of these guide RNPs reside in nucleoplasmic dots, called Cajal bodies, which are transited by snRNPs during their biogenesis. We identified a protein responsible for localization of these guide RNPs, as well as telomerase, in Cajal bodies.

Our studies of the biogenesis of microRNAs have revealed that the earliest steps of processing of the primary microRNA transcript occur preferentially before release from the chromatin template. We have also investigated the roles of post-translational modifications and of the stoichiometry of Drosha and DGCR8, components of the Microprocessor essential for this nuclear processing step. Under conditions of cell cycle arrest, which induce a state called quiescence, the efficiency of protein synthesis actually increases rather than decreases in the presence of microRNAs targeting the 3′-UTR of mRNAs, apparently due to different proteins (in addition to Argonaut) associating with the microRNPs.

Deciphering the roles of viral noncoding RNPs found in primate cells infected by gamma-herpesviruses is a challenging problem. This family of viruses is characterized by both lytic (replicative) and latent states. We recently found that viral snRNPs made during latent infection by Herpesvirus saimiri, an agent that causes malignant transformation of the T cells of the immune system of monkeys, are involved in up-regulation of genes in the T-cell activation pathway. The molecular mechanism involves the binding and degradation of a specific host microRNA. Our discovery of snRNA-microRNA chimeric transcripts in the same virus has led us to uncover a novel microRNA biogenesis pathway in mammalian cells, where the pre-microRNAs initiate with a cap structure. Epstein-Barr virus (EBV), which causes mononucleosis as well as certain human cancers, encodes two small RNAs called EBERs that are extremely abundant in the nucleus of transformed cells.  We discovered that one of the EBERs interacts with the terminal repeat regions of the EBV genome and contributes to viral DNA replication, which is linked to EBV oncogenesis.

Investigation of another abundant viral noncoding RNP called PAN (for polyadenylated nuclear), which is present in cells lytically infected by Kaposi sarcoma–associated herpesvirus (KSHV—the cause of the most common cancer afflicting AIDS patients) and is required for late viral protein synthesis, has revealed that it contains an RNA element (called the ENE) responsible for its high nuclear accumulation. We solved the x-ray structure of the ENE bound to oligo(A)9, revealing an elegant triple-stranded structure that clamps the poly(A) tail of PAN RNA. These structural insights into the exact interaction between the ENE and poly(A) allowed identification of ENE-like structures in other viral and cellular noncoding RNAs. Recently, solving the x-ray structure of an ENE from MALAT1 (metastasis-associated lung adenocarcinoma transcript), an abundant vertebrate noncoding RNA, has provided an explanation for why triple helices longer than 6 have not been observed in naturally-occurring RNAs.

As of December 15, 2014

Scientist Profile

Investigator
Yale University
Biochemistry, Molecular Biology