X-chromosome kiss and tell: how the Xs go their separate ways.

Loci associated with noncoding RNAs have important roles in X-chromosome inactivation (XCI), the dosage compensation mechanism by which one of two X chromosomes in female cells becomes transcriptionally silenced. The Xs start out as epigenetically equivalent chromosomes, but XCI requires a cell to treat two identical X chromosomes in completely different ways: One X chromosome must remain transcriptionally active while the other becomes repressed. In the embryo of eutherian mammals, the choice to inactivate the maternal or paternal X chromosome is random. The fact that the Xs always adopt opposite fates hints at the existence of a trans-sensing mechanism to ensure the mutually exclusive silencing of one of the two Xs. This paper highlights recent evidence supporting a model for mutually exclusive choice that involves homologous chromosome pairing and the placement of asymmetric chromatin marks on the two Xs.

Lack of catalytic activity of a murine mRNA cytoplasmic serine hydroxymethyltransferase splice variant: evidence against alternative splicing as a regulatory mechanism.

Mammalian serine hydroxymethyltransferase (SHMT) is a tetrameric, pyridoxal phosphate-dependent enzyme that catalyzes the reversible interconversion of serine and tetrahydrofolate to glycine and methylenetetrahydrofolate. This reaction generates single-carbon units for purine, thymidine, and methionine biosynthesis. Cytoplasmic SHMT (cSHMT) has been postulated to channel one-carbon substituted folates to various folate-dependent enzymes, and alternative splicing of the cSHMT transcript may be a mechanism that enables specific protein-protein interactions. The cytoplasmic isozyme is expressed from species-specific and tissue-specific alternatively spliced transcripts that encode proteins with modified carboxy-terminal domains, while the mitochondrial isozyme is expressed from a single transcript. While the full-length mouse and human cSHMT proteins are 91% identical, their alternatively spliced transcripts differ. The murine cSHMT gene is expressed as two transcripts. One transcript encodes a full-length 55 kDa active enzyme (cSHMT), while the other transcript encodes a 35 kDa protein (McSHMTtr). The McSHMTtr protein present in mouse liver and kidney does not bind 5-formyltetrahydrofolate, nor does it oligomerize with the full-length cSHMT enzyme. While recombinant cSHMT-glutathione S-transferase fusion proteins form tetramers and are catalytically active, McSHMTtr-glutathione S-transferase fusion proteins are catalytically inactive, do not form heterotetramers, and do not bind pyridoxal phosphate. Analysis of the murine cSHMT crystal structure indicates that the active site lysine that normally binds pyridoxal phosphate in the cSHMT protein is exposed to solvent in the McSHMTtr protein, preventing stable formation of a Schiff base with pyridoxal phosphate. Modeling studies suggest that the human cSHMT proteins expressed from alternatively spliced transcripts are inactive as well. Therefore, channeling mechanisms enabling specific protein-protein interactions of active enzymes are not based on cSHMT alternative splicing.

Polyadenylation of three classes of chloroplast RNA in Chlamydomonas reinhadtii.

Three classes of RNA, represented by atpB and petD mRNAs, Arg and Glu tRNAs, and 5S rRNA, were found to exist in polyadenylated form in Chlamydomonas reinhardtii chloroplasts. Sequence analysis of cDNA clones derived from reverse transcriptase-polymerase chain reaction protocols used to select polyadenylated RNAs revealed that, at least for the mRNAs and tRNAs, there are three apparent types of polyadenylation. In the first case, the poly(A) tail is added at or near the mature 3' end, even when this follows a strong secondary structure. In the second case, the tail is added to pre-mRNA or pre-tRNA, suggesting a possible competition between polyadenylation and RNA-processing pathways. Finally, in all cases, the poly(A) tail can be added internally, possibly as a part of an RNA-decay pathway. The tails found in Chlamydomonas chloroplasts differ from those of spinach chloroplasts in adenine content, being nearly homopolymeric (>98% adenine) versus 70% in spinach, and are similar in length to those of Escherichia coli, being mostly between 20 and 50 nt. In vitro assays using a Chlamydomonas chloroplast protein extract showed that a 3' end A25 tail was sufficient to stimulate rapid degradation of atpB RNA in vitro, with a lesser effect for petD, and only minor effects on trnE. We therefore propose that polyadenylation contributes to mRNA degradation in Chlamydomonas chloroplasts, but that its effect may vary.