Serum microRNA expression as an early marker for breast cancer risk in prospectively collected samples from the Sister Study cohort.

INTRODUCTION: MicroRNAs (miRNAs) are small, non-coding, single-stranded RNAs between 18-22 nucleotides long that regulate gene expression. Expression of miRNAs is altered in tumor compared to normal tissue; there is some evidence that these changes may be reflected in the serum of cancer cases compared to healthy individuals. This has yet to be examined in a prospective study where samples are collected before diagnosis. METHODS: We used Affymetrix arrays to examine serum miRNA expression profiles in 410 participants in the Sister Study, a prospective cohort study of 50,884 women. All women in the cohort had never been diagnosed with breast cancer at the time of enrollment. We compared global miRNA expression patterns in 205 women who subsequently developed breast cancer and 205 women who remained breast cancer-free. In addition within the case group we examined the association of miRNA expression in serum with different tumor characteristics, including hormone status (ER, PR, and HER-2) and lymph node status. RESULTS: Overall, 414 of 1,105 of the human miRNAs on the chip were expressed above background levels in 50 or more women. When the average expression among controls was compared to cases using conditional logistic regression, 21 miRNAs were found to be differentially expressed (P≤.05). Using qRT-PCR on a small, independent sample of 5 cases and 5 controls we verified overexpression of the 3 highest expressing miRNAs among cases, miR-18a, miR-181a, and miR-222; the differences were not statistically significant in this small set. The 21 differentially expressed miRNAs are known to target at least 82 genes; using the gene list for pathway analysis we found enrichment of genes involved in cancer-related processes. In a separate case-case analyses restricted to the 21 miRNAs, we found 7 miRNAs with differential expression for women whose breast tumors differed by HER-2 expression, and 10 miRNAs with differential expression by nodal status. CONCLUSIONS: miRNA levels in serum show a number of small differences between women who later develop cancer versus those who remain cancer-free.

Interaction between FLASH and Lsm11 is essential for histone pre-mRNA processing in vivo in Drosophila.

Metazoan replication-dependent histone mRNAs are the only nonpolyadenylated cellular mRNAs. Formation of the histone mRNA 3' end requires the U7 snRNP, which contains Lsm10 and Lsm11, and FLASH, a processing factor that binds Lsm11. Here, we identify sequences in Drosophila FLASH (dFLASH) that bind Drosophila Lsm11 (dLsm11), allow localization of dFLASH to the nucleus and histone locus body (HLB), and participate in histone pre-mRNA processing in vivo. Amino acids 105-154 of dFLASH bind to amino acids 1-78 of dLsm11. A two-amino acid mutation of dLsm11 that prevents dFLASH binding but does not affect localization of U7 snRNP to the HLB cannot rescue the lethality or histone pre-mRNA processing defects resulting from an Lsm11 null mutation. The last 45 amino acids of FLASH are required for efficient localization to the HLB in Drosophila cultured cells. Removing the first 64 amino acids of FLASH has no effect on processing in vivo. Removal of 13 additional amino acids of dFLASH results in a dominant negative protein that binds Lsm11 but inhibits processing of histone pre-mRNA in vivo. Inhibition requires the Lsm11 binding site, suggesting that the mutant dFLASH protein sequesters the U7 snRNP in an inactive complex and that residues between 64 and 77 of dFLASH interact with a factor required for processing. Together, these studies demonstrate that direct interaction between dFLASH and dLsm11 is essential for histone pre-mRNA processing in vivo and for proper development and viability in flies.

The Drosophila U7 snRNP proteins Lsm10 and Lsm11 are required for histone pre-mRNA processing and play an essential role in development.

Metazoan replication-dependent histone mRNAs are not polyadenylated, and instead terminate in a conserved stem-loop structure generated by an endonucleolytic cleavage of the pre-mRNA involving U7 snRNP. U7 snRNP contains two like-Sm proteins, Lsm10 and Lsm11, which replace SmD1 and SmD2 in the canonical heptameric Sm protein ring that binds spliceosomal snRNAs. Here we show that mutations in either the Drosophila Lsm10 or the Lsm11 gene disrupt normal histone pre-mRNA processing, resulting in production of poly(A)+ histone mRNA as a result of transcriptional read-through to cryptic polyadenylation sites present downstream from each histone gene. This molecular phenotype is indistinguishable from that which we previously described for mutations in U7 snRNA. Lsm10 protein fails to accumulate in Lsm11 mutants, suggesting that a pool of Lsm10-Lsm11 dimers provides precursors for U7 snRNP assembly. Unexpectedly, U7 snRNA was detected in Lsm11 and Lsm1 mutants and could be precipitated with anti-trimethylguanosine antibodies, suggesting that it assembles into a snRNP particle in the absence of Lsm10 and Lsm11. However, this U7 snRNA could not be detected at the histone locus body, suggesting that Lsm10 and Lsm11 are necessary for U7 snRNP localization. In contrast to U7 snRNA null mutants, which are viable, Lsm10 and Lsm11 mutants do not survive to adulthood. Because we cannot detect differences in the histone mRNA phenotype between Lsm10 or Lsm11 and U7 mutants, we propose that the different terminal developmental phenotypes result from the participation of Lsm10 and Lsm11 in an essential function that is distinct from histone pre-mRNA processing and that is independent of U7 snRNA.

A genome-wide RNA interference screen reveals that variant histones are necessary for replication-dependent histone pre-mRNA processing.

Metazoan replication-dependent histone mRNAs are not polyadenylated and instead end in a conserved stem loop that is the cis element responsible for coordinate posttranscriptional regulation of these mRNAs. Using biochemical approaches, only a limited number of factors required for cleavage of histone pre-mRNA have been identified. We therefore performed a genome-wide RNA interference screen in Drosophila cells using a GFP reporter that is expressed only when histone pre-mRNA processing is disrupted. Four of the 24 genes identified encode proteins also necessary for cleavage/polyadenylation, indicating mechanistic conservation in formation of different mRNA 3' ends. We also unexpectedly identified the histone variants H2Av and H3.3A/B. In H2Av mutant cells, U7 snRNP remains active but fails to accumulate at the histone locus, suggesting there is a regulatory pathway that coordinates the production of variant and canonical histones that acts via localization of essential histone pre-mRNA processing factors.

U7 snRNA mutations in Drosophila block histone pre-mRNA processing and disrupt oogenesis.

Metazoan replication-dependent histone mRNAs are not polyadenylated, and instead terminate in a conserved stem-loop structure generated by an endonucleolytic cleavage involving the U7 snRNP, which interacts with histone pre-mRNAs through base-pairing between U7 snRNA and a purine-rich sequence in the pre-mRNA located downstream of the cleavage site. Here we generate null mutations of the single Drosophila U7 gene and demonstrate that U7 snRNA is required in vivo for processing all replication-associated histone pre-mRNAs. Mutation of U7 results in the production of poly A+ histone mRNA in both proliferating and endocycling cells because of read-through to cryptic polyadenylation sites found downstream of each Drosophila histone gene. A similar molecular phenotype also results from mutation of Slbp, which encodes the protein that binds the histone mRNA 3' stem-loop. U7 null mutants develop into sterile males and females, and these females display defects during oogenesis similar to germ line clones of Slbp null cells. In contrast to U7 mutants, Slbp null mutations cause lethality. This may reflect a later onset of the histone pre-mRNA processing defect in U7 mutants compared to Slbp mutants, due to maternal stores of U7 snRNA. A double mutant combination of a viable, hypomorphic Slbp allele and a viable U7 null allele is lethal, and these double mutants express polyadenylated histone mRNAs earlier in development than either single mutant. These data suggest that SLBP and U7 snRNP cooperate in the production of histone mRNA in vivo, and that disruption of histone pre-mRNA processing is detrimental to development.

The Chlamydomonas reinhardtii proteins Ccp1 and Ccp2 are required for long-term growth, but are not necessary for efficient photosynthesis, in a low-CO2 environment.

The unicellular green alga Chlamydomonas reinhardtii acclimates to a low-CO2 environment by modifying the expression of a number of messages. Many of the genes that increase in abundance during acclimation to low-CO2 are under the control of the putative transcription factor Cia5. C. reinhardtii mutants null for cia5 do not express several of the known low-CO2 inducible genes and do not grow in a low-CO2 environment. Two of the genes under the control of Cia5, Ccp1 and Ccp2 , encode polypeptides that are localized to the chloroplast envelope and have a high degree of similarity to members of the mitochondrial carrier family of proteins. Since their discovery, Ccp1/2 have been candidates for bicarbonate uptake proteins of the chloroplast envelope membrane. In this report, RNA interference was successful in dramatically decreasing the abundance of the mRNAs for Ccp1 and Ccp2 . The abundance of the Ccp1 and Ccp2 proteins were also reduced in the RNAi strains. The RNAi strains grew slower than WT in a low-CO2 environment, but did not exhibit a mutant carbon concentrating phenotype as determined by the cells' apparent affinity for dissolved inorganic carbon. Possible explanations of this RNAi phenotype are discussed.

Rubisco activase is required for optimal photosynthesis in the green alga Chlamydomonas reinhardtii in a low-CO(2) atmosphere.

This report describes a Chlamydomonas reinhardtii mutant that lacks Rubisco activase (Rca). Using the BleR (bleomycin resistance) gene as a positive selectable marker for nuclear transformation, an insertional mutagenesis screen was performed to select for cells that required a high-CO2 atmosphere for optimal growth. The DNA flanking the BleR insert of one of the high-CO2-requiring strains was cloned using thermal asymmetric interlaced-polymerase chain reaction and inverse polymerase chain reaction and sequenced. The flanking sequence matched the C. reinhardtii Rca cDNA sequence previously deposited in the National Center for Biotechnology Information database. The loss of a functional Rca in the strain was confirmed by the absence of Rca mRNA and protein. The open reading frame for Rca was cloned and expressed in pSL18, a C. reinhardtii expression vector conferring paromomycin resistance. This construct partially complemented the mutant phenotype, supporting the hypothesis that the loss of Rca was the reason the mutant grew poorly in a low-CO2 atmosphere. Sequencing of the C. reinhardtii Rca gene revealed that it contains 10 exons ranging in size from 18 to 470 bp. Low-CO2-grown rca1 cultures had a growth rate and maximum rate of photosynthesis 60% of wild-type cells. Results obtained from experiments on a cia5 rca1 double mutant also suggest that the CO2-concentrating mechanism partially compensates for the absence of an active Rca in the green alga C. reinhardtii.

Use of the bleomycin resistance gene to generate tagged insertional mutants of Chlamydomonas reinhardtii that require elevated CO2 for optimal growth.

Chlamydomonas reinhardtii Dangeard possesses a CO2 concentrating mechanism (CCM) that enables it to grow at very low CO2 concentrations. In previous studies, insertional mutagenesis was successfully used to identify genes required for growth at low CO2 in C. reinhardtii. These earlier studies used the C. reinhardtii genes, Nit1 and Arg7 to complement nit1- or arg7- strains, thereby randomly inserting a second copy of Nit1 or Arg7 into the genome. Because these genes are already present in the C. reinhardtii genome, it was often difficult to identify the location of the inserted DNA and the gene disrupted by the insertion. We have developed a transformation protocol using the BleR gene, which confers resistance to the antibiotic Zeocin. The insertion of this gene allows one to use a variety of existing polymerase chain reaction (PCR) methodologies to identify the disrupted gene. In this study the D66 strain (nit2-, cw15, mt+) was transformed by electroporation using a plasmid containing the BleR gene. Primary transformants (42 000) were obtained after growth in the dark on acetate plus Zeocin medium. Colonies were then tested for their ability to grow photosynthetically on elevated CO2 or low levels of CO2 (100 ppm). About 120 mutants were identified which grew on elevated CO2 but were unable to grow well at low CO2 concentrations. About 50% of these mutants had low affinities for inorganic carbon as assessed by K0.5(CO2), indicating a potential defect in the CCM. The location of the inserted DNA is being determined using inverse PCR (iPCR) and thermal asymmetric interlaced (TAIL) PCR. Using these methods, one can rapidly locate the inserted DNA in the genome and identify the gene that has been disrupted by the insertion.