Fluorescent in situ hybridization of ovaries of the indicated genotypes using an H3 coding probe or a probe (H3-ds) that only detects mis-processed or read through transcripts Note that

RNA pol II: embryo 14-16h

WT

PBac/Df

FL

1-733

merge

merge

coding

4x mag.

B

A

mxc

RNA pol II: Kc167 cells

Input: embryo 14-16h Input: Kc167 cells 0-8868 0-69292 0-1434 0-2906

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histone H3 mRNA accumulates only during S-phase and that the nurse cell endocycles are asynchronous. The cytoplasmic H3-ds signal in FLASHPBac/Df _{represents mis-processed H3} transcripts that were polyadenylated and exported. The small amounts of misprocessed RNA in FLASH1-733 and mxcG46 (see Fig. 5B) are below our threshold of detection. The nuclear foci in FLASH1-733 and mxcG46 mutants detected with the H3-ds probe (yellow arrows) are nascent transcripts at the histone locus. Scale bar = 10 µm.

DISCUSSION

The HLB Contributes to Multiple Steps in Histone mRNA Biosynthesis

We previously showed that a sequence in, and activity from, the H3-H4 promoter promotes stepwise HLB assembly and coordinate transcriptional activation of the four core histone genes (Salzler et al., 2013). Here we show that the HLB also functions to ensure proper histone mRNA 3’ end formation by concentrating pre-mRNA processing factors at the histone locus. FLASH and U7 snRNP accumulation in the HLB does not require any cis elements in histone pre- mRNA, and they persist in the HLB in the absence of histone gene transcription (Salzler et al., 2013; White et al., 2011). Our data indicate that the HLB mediates rapid histone pre-mRNA cleavage and subsequent transcription termination, providing a clear example of how a nuclear body contributes to efficient gene expression.

Concentrating Factors within the HLB Ensures Efficient Histone mRNA Synthesis. In this and our previous studies, we show that mis-processed, polyadenylated histone mRNAs accumulate i) when genes encoding components of the histone pre-mRNA processing

machinery (e.g. FLASH, Slbp or U7 snRNP) are mutated (Godfrey et al., 2006; Godfrey et al., 2009; Lanzotti et al., 2002; Sullivan et al., 2001) ii) after engineering mutations in FLASH that interfere with Lsm11 binding or impair recruitment of the HCC, and iii) by preventing

concentration in the HLB of a FLASH protein that is biochemically impaired for processing. Whether the cryptic poly A signals encoded after the HDE in each Drosophila histone gene provide an important function is not known. Nevertheless, our data suggest that one role of the

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HLB is to promote a high concentration of pre-mRNA processing factors that facilitate stem loop- and HDE-mediated 3’ end formation rather than polyadenylation.

When FLASH and/or U7 snRNP are present at normal levels but not concentrated in the HLB, or when the biochemically attenuated FLASHLDIY71 is localized in the HLB, we observe by in situ hybridization the accumulation of longer, nascent transcripts that extend past the normal processing site. The appearance of these longer, nascent transcripts indicates that normal processing is altered. We propose that the rate of cleavage has slowed resulting in failure of polymerase dissociation and continued transcription. Thus, by increasing the local concentration of processing activity, the HLB affects the rate of histone pre-mRNA processing. Interestingly, S1 analysis indicates that most of these RNAs are processed at the normal site, and only a small amount is polyadenylated. This normal processing requires FLASH, since severely depleting FLASH by mutation results in nearly 100% conversion to polyadenylated histone mRNA.

What is the basis for these RNA phenotypes? Mechanistic differences between

processing 3’ ends of polyadenylated mRNAs and histone mRNAs may provide an answer. The interaction of polyadenylation factors with the CTD of RNA pol II tightly couples polyadenylation with transcription (Adamson et al., 2005; Hirose and Manley, 1998; Proudfoot, 2004). In

contrast, an interaction between RNA pol II and the histone-processing complex is not

necessary for histone mRNA 3’ end formation in vitro (Adamson and Price, 2003). This result suggests that processing at the normal cleavage site can occur after the polymerase has transcribed well past the HDE, making histone pre-mRNA processing more flexible than processing of polyadenylated mRNAs (Figure 3.10).

A second possible way to accumulate properly processed histone mRNA from longer transcripts is to “reprocess” a transcript that has already been either cleaved or polyadenylated (Pandey et al., 1994). In fact, in C. elegans the proposed normal pathway of histone mRNA processing is polyadenylation followed by cleavage after the stem loop by an siRNA-like

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mechanism (Avgousti et al., 2012; Mangone et al., 2010). Thus, when normal processing is slowed, the histone pre-mRNA processing machinery may be able to act on different RNA substrates generated by continued transcription past the HDE, resulting in cleavage at the normal location (Figure 3.10).

Our analysis of publically available data of RNA pol II occupancy on Drosophila histone genes suggests that transcription terminates close to the HDE. Similar results were found in mammalian cells suggesting that this is a general property of histone genes (Anamika et al., 2012; Cheng et al., 2012). In Drosophila, strong RNA polymerase II transcriptional pause sites just past the HDE were detected biochemically (Adamson and Price, 2003). Polymerase pausing may promote pre-mRNA processing followed by rapid, subsequent transcription termination. We propose that the HLB contributes to the coupling of histone 3’ end formation and transcription termination by accelerating histone pre-mRNA cleavage.

Multiple Domains of FLASH are Required for Rapid Histone pre-mRNA Processing. Our studies indicate that FLASH contributes to normal histone mRNA 3’ end formation in several distinct ways. Interaction of FLASH with another HLB component, Lsm11, and

subsequent recruitment of the HCC is required for pre-mRNA processing (Sabath et al., 2013). As shown here, concentrating FLASH in the HLB is also required for recruitment of U7 snRNP to the HLB, an activity distinct from interaction of FLASH with Lsm11 required for pre-mRNA processing. Biochemical studies with both Drosophila and HeLa cell extracts suggest that the active form of U7 snRNP is assembled with FLASH and the HCC prior to interacting with histone pre-mRNA (Sabath et al., 2013; Yang et al., 2013). Thus, the interaction of FLASH and Lsm11 to form an active U7 cleavage complex may be regulated within the HLB independently of recruiting these factors to the HLB.

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Cellular Microenvironments that Enhance Biological Processes

Our results support the idea that NBs facilitate reactions by concentrating factors in a particular sub-domain of the nucleus (Mao et al., 2011; Matera et al., 2009). The exchange of NB

components with the adjacent environment is slower than expected by diffusion, promoting concentration in NBs (Dundr et al., 2004). Such concentration of components creates discrete micro-domains with different physical chemical properties than the surrounding nucleoplasm that could facilitate biological processes (Brangwynne et al., 2011). In the case of the HLB, our data provides evidence for two roles: enhancing the rate of pre-mRNA processing and

promoting coupling of steps in histone mRNA synthesis. RNA binding proteins with low complexity domains assemble into bodies in vivo and can form a gel in solution, which may mimic some properties of nuclear bodies (Han et al., 2012; Kato et al., 2012; Lee et al., 2013). FLASH and Mxc are large proteins that contain low complexity domains that may promote the formation of the HLB through association with other proteins.

The cytoplasm also contains distinct non-membrane bound microenvironments similar to NBs. While the majority of examples involve RNA regulation (e.g. processing bodies, stress granules, P bodies (Decker et al., 2007; Brangwynne et al., 2009; Wippich et al., 2013) other structures such as the purinosome, an environment containing a concentration of the six enzymes involved in de novo purine synthesis (An et al., 2008), lipid rafts and the centrosome (Decker et al., 2011) each contain high concentrations of factors involved in a distinct process. Increased understanding of how NBs assemble and contribute to biological reactions will likely translate to understanding other macromolecular complexes and microenvironments that function in other cellular compartments.

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Figure 3.10

Figure 3.10 Model of HLB participation in histone pre-mRNA processing

A. SLBP (orange) interacts directly with the stem loop (SL) in the pre-mRNA downstream of the